Youngshim Lee

Anticancer and structure-activity relationship evaluation of 3-(naphthalen-2-yl)-N,5-diphenyl-pyrazoline-1-carbothioamide analogs of chalcone.

To identify new potent chemotherapeutic agents, we synthesized compounds with 3-(naphthalen-2-yl)-N,5-diphenyl-pyrazoline-1-carbothioamide (NDPC) skeletons and evaluated their cytotoxicities using a clonogenic long-term survival assay. Their half-maximal cell growth inhibitory concentrations ranged from a few hundred nanomolars to a few micromolars. Further biological experiments including flow cytometry and western blotting analysis were performed with the derivative showing the best cytotoxicity. To identify a target protein of the selected compound, an in vitro kinase assay was carried out, which revealed that aurora kinases A and B were inhibited by the test compound, and this was confirmed using western blot analysis. The molecular binding mode between the selected compound and the kinases was elucidated using in silico docking. The structural conditions required for good cytotoxicity were identified based on the quantitative relationships between the physicochemical properties of the derivatives and their cytotoxicities.

Production of Three O-Methhylated Esculetins with Escherichia coli Expressing O-Methyltransferase from Poplar

O-Methyltransferase, POMT-9 was expressed in Escherichia coli. HPLC analysis of reaction products revealed three peaks corresponding to isoscopoletin, scopoletin, and scoparone, and their structures were determined using NMR. Biotransformation of esculetin with E. coli expressing POMT-9 generated scopoletin, isoscopoletin, and scoparone at 30.3, 21, and 31 μM respectively. POMT-9 is the first O-methyltransferase that produces three different O-methylated products.

Arsenic bioavailability in soils before and after soil washing: the use of Escherichia coli whole-cell bioreporters

We investigated the quantification of bioavailable arsenic in contaminated soils and evaluation of soil-washing processes in the aspect of bioavailability using a novel bacterial bioreporter developed in present study. The whole-cell bioreporter (WCB) was genetically engineered by fusing the promoter of nik operon from Escherichia coli and green fluorescent protein as a sensing domain and reporter domain. Among eight well-known hazardous heavy metals and metalloid, this system responded specifically to arsenic, thereby inferring association of As(III) with NikR inhibits the repression. Moreover, the response was proportional to the concentration of As(III), thereby it was capable to determine the amount of bioavailable arsenic quantitatively in contaminated soils. The bioavailable portion of arsenic was 5.9 (3.46–10.96) and 0.9 (0.27–1.74) % of total from amended and site soils, respectively, suggesting the bioavailability of arsenic in soils was related to the soil properties and duration of aging. On the other hand, only 1.37 (0.21–2.97) % of total arsenic was extracted into soil solutions and 19.88 (11.86–28.27) % of arsenic in soil solution was bioavailable. This result showed that the soluble arsenic is not all bioavailable and most of bioavailable arsenic in soils is water non-extractable. In addition, the bioavailable arsenic was increased after soil-washing while total amount was decreased, thereby suggesting the soil-washing processes release arsenic associated with soil materials to be bioavailable. Therefore, it would be valuable to have a tool to assess bioavailability and the bioavailability should be taken into consideration for soil remediation plans.

Use of Tunable Whole-Cell Bioreporters to Assess Bioavailable Cadmium and Remediation Performance in Soils

It is important to have tools to measure the bioavailability to assess the risks of pollutants because the bioavailability is defined as the portions of pollutants showing the biological effects on living organisms. This study described the construction of tunable Escherichia coli whole-cell bioreporter (WCB) using the promoter region of zinc-inducible operon and its application on contaminated soils. It was verified that this WCB system showed specific and sensitive responses to cadmium rather than zinc in the experimental conditions. It was inferred that Cd(II) associates stronger with ZntR, a regulatory protein of zinc-inducible operon, than other metal ions. Moreover, the expression of reporter genes, egfp and mcherry, were proportional to the concentration of cadmium, thereby being a quantitative sensor to monitor bioavailable cadmium. The capability to determine bioavailable cadmium was verified with Cd(II) amended LUFA soils, and then the applicability on environmental systems was investigated with field soils collected from smelter area in Korea before and after soil-washing. The total amount of cadmium was decreased after soil washing, while the bioavailability was increased. Consequently, it would be valuable to have tools to assess bioavailability and the effectiveness of soil remediation should be evaluated in the aspect of bioavailability as well as removal efficiency.

Biological Synthesis of Baicalein Derivatives Using Escherichia coli.

Two baicalein derivatives, baicalin and oroxylin A, were synthesized in this study. These derivatives exhibit diverse biological activities, such as anxiolytic and anticancer activities as well as memory enhancement. In order to synthesize baicalin from aglycon baicalein using Escherichia coli, we utilized a glycosyltransferase that regioselectively transfers glucuronic acid from UDP-glucuronic acid to the 7-hydroxy group of baicalein. To increase baicalin productivity, an araA deletion E. coli mutant, which accumulates UDP-glucuronic acid, was used, and ugd, which converts UDP-glucose to UDP-glucuronic acid, was overexpressed. Using these strategies, approximately 720.3 µM baicalin was synthesized from 1,000 µM baicalein. Oroxylin A was then synthesized from baicalein. Two O-methyltransferases (OMTs), ROMT-15 and POMT-9, were tested to examine the production of oroxylin A from baicalein. E. coli harboring ROMT-15 and E. coli harboring POMT-9 produced reaction products that had different retention times, indicating that they are methylated at different positions; the structure of the reaction product from POMT-9 was consistent with oroxylin A, whereas that from ROMT-15 was 7-O-methyl baicalein. Using E. coli harboring POMT-9, approximately 50.3 mg/l of oroxylin A (177 µM) was synthesized from 54 mg/l baicalein (200 µM).

The Use of Reductive Methylation of Lysine Residues to Study Protein-Protein Interactions in High Molecular Weight Complexes by Solution NMR

While solution state NMR is very well suited for analysis of protein-protein interactions occurring with a wide range of affinities, it suffers from one significant weakness, known as the molecular weight limitation. This limitation stems from the efficient nuclear relaxation processes in macromolecules larger than 30 kDa (Wider & Wuthrich, 1999). These relaxation processes cause rapid decay of NMR signals. Although the use of transverse relaxation optimized spectroscopy (TROSY) approaches has made solution state NMR of large proteins and protein-protein complexes more feasible, it is still limited by the ability to produce isotope enriched proteins (Pervushin et al., 1997). However, there is a significant number of proteins for which no convenient system for stable isotope incorporation exists. We recently utilized reductive methylation methodology to demonstrate that it is possible to introduce 13C-enriched methyl groups into lysine residues in otherwise unlabeled proteins with the purpose of studying protein-ligand and protein-protein interactions by NMR (Abraham et al., 2008).

Biased antagonism of CXCR4 avoids antagonist tolerance

Selectively blocking G protein signaling but not GPCR internalization may provide therapeutic benefit. The right kind of bias AMD3100, an antagonist of the chemokine receptor CXCR4, prevents the accumulation of leukemic cells in the bone marrow, which promotes the efficacy of chemotherapeutic agents. However, tolerance to AMD3100 can develop, which leads to receptor accumulation on the cell surface and retention of cells in the bone marrow. Hitchinson et al. showed that a peptide derived from CXCR4 avoided the development of tolerance by acting as a biased antagonist of G protein signaling but not β-arrestin–mediated internalization of CXCR4. Similar properties were shared by a nonpeptide, small-molecule inhibitor that also did not stimulate tolerance. These results suggest that the use of biased GPCR antagonists could be of therapeutic benefit in patients who have developed tolerance to nonbiased antagonists. Repeated dosing of drugs targeting G protein–coupled receptors can stimulate antagonist tolerance, which reduces their efficacy; thus, strategies to avoid tolerance are needed. The efficacy of AMD3100, a competitive antagonist of the chemokine receptor CXCR4 that mobilizes leukemic blasts from the bone marrow into the blood to sensitize them to chemotherapy, is reduced after prolonged treatment. Tolerance to AMD3100 increases the abundance of CXCR4 on the surface of leukemic blasts, which promotes their rehoming to the bone marrow. AMD3100 inhibits both G protein signaling by CXCR4 and β-arrestin1/2–dependent receptor endocytosis. We demonstrated that biased antagonists of G protein–dependent chemotaxis but not β-arrestin1/2 recruitment and subsequent receptor endocytosis avoided tolerance. The peptide antagonist X4-2-6, which is derived from transmembrane helix 2 and extracellular loop 1 of CXCR4, limited chemotaxis and signaling but did not promote CXCR4 accumulation on the cell surface or cause tolerance. The activity of X4-2-6 was due to its distinct mechanism of inhibition of CXCR4. The peptide formed a ternary complex with the receptor and its ligand, the chemokine CXCL12. Within this complex, X4-2-6 released the portion of CXCL12 critical for receptor-mediated activation of G proteins but enabled the rest of the chemokine to recruit β-arrestins to the receptor. In contrast, AMD3100 displaced all components of the chemokine responsible for CXCR4 activation. We further identified a small molecule with similar biased antagonist properties to those of X4-2-6, which may provide a viable alternative to patients when antagonist tolerance prevents drugs from reaching efficacy.

Synthetic Diethyl 2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylates Induce Apoptosis

BACKGROUND The Hantzsch ester, diethyl 2,6-dimethyl-1,4-dihydropyridine-3,5- dicarboxylate, has been used as a hydride donor and its various biological effects have been reported. To identify chemotherapeutic agents with apoptotic effects, 21 diethyl 2,6-dimethyl-1,4- dihydropyridine-3,5-dicarboxylates were designed and synthesized; they have not been reported as apoptosis inducers thus far. Their structure-cytotoxicity relationships were investigated. Further biological experiments were performed on the title compound. METHODS The cytotoxicities of the current synthetic compounds were measured using a clonogenic assay in HCT116 human colon cancer cells. An annexin V staining assay was used to confirm if the title compound induced apoptosis. To identify the synthetic compounds, Nuclear Magnetic Resonance (NMR) spectroscopy and high-resolution mass spectrometry (HR-MS) were conducted. As molecular symmetry was observed in the NMR spectroscopic data, the three dimensional structures were determined from ab initio calculations and X-ray crystallography. RESULTS The results obtained from NMR spectroscopy, ab initio calculations, and X-ray crystallography revealed that the diethyl 2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate derivatives synthesized in this research have symmetric structures. The cytotoxicities of the 21 derivatives were tested in the HCT116 human colon cancer cell lines, and their half-maximal cell growth inhibitory concentrations ranged between 16.29 and 68.88 µM. Structure-cytotoxicity relationships demonstrated that bulky substitutions were preferred, para-positioned substituents tended to have better cytotoxic values, and the polarity may have a function as well. The cytotoxicity of the title compound in HCT116 colon cancer cells was mediated through apoptotic cell death. CONCLUSION To obtain chemotherapeutic agents that induce apoptosis, 21 diethyl 2,6-dimethyl- 1,4-dihydropyridine-3,5-dicarboxylates were designed and synthesized. NMR spectroscopy, ab initio calculations, and X-ray crystallography demonstrated that the diethyl 2,6-dimethyl-1,4- dihydropyridine-3,5-dicarboxylate derivatives synthesized in this research had symmetric structures. Even if the half-maximal cell growth inhibitory concentrations of the 21 derivatives did not show dramatic inhibitory activity against HCT116 human colon cancer cells, small changes in the structure affected the anticancer activities. Treatment with diethyl 4-(4-chlorophenyl)-2,6- dimethyl-1,4-dihydropyridine-3,5-dicarboxylate substantially reduced the cell viability and the cytotoxicity against HCT116 colon cancer cells was mediated through apoptotic cell death. As the ability of diethyl 2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylates to induce apoptosis has not been previously reported, we have now reported their design, synthesis, cytotoxicity, and structureactivity relationships.

Cell growth inhibitory effects of polyphenols with naphthalene skeleton against cisplatin-resistant ovarian cancer cells

Cisplatin often shows the drug resistance which could limit the chemotherapeutic efficacy. Thus, it is necessary to develop anticancer agents against cisplatin-resistant cancer cells. To identify pharmacophores exhibiting the cell growth inhibitory effect against cisplatin-resistant A2780/Cis ovarian cancer cells, we prepared 35 synthetic polyphenols bearing naphthalene skeleton including naphthalenyl chalcones, naphthalenyl flavones, naphthalenyl flavanones, 4,5-dihydro-1H-pyrazol-3-yl)naphthalen-2-ols, naphthalen-1-yl-N-phenyl-4,5-dihydro-1H-pyrazole-1-carbothioamides, and 4,5-dihydro-1H-pyrazol-3-yl)naphthalen-1-ol. The correlation between their inhibitory effects and structural properties was evaluated using hologram quantitative structure activity relationship and comparative molecular field analysis. The pharmacophores derived here can lead us to design new polyphenols against the growth of cisplatin-resistant cells.

Inhibitory potential of flavonoids on PtdIns(3,4,5)P3 binding with the phosphoinositide-dependent kinase 1 pleckstrin homology domain.

Many membrane-associated proteins are involved in various signaling pathways, including the phosphoinositide 3-kinase (PI3K) pathway, which has key roles in diverse cellular processes. Disruption of the activities of these proteins is involved in the development of disease in humans, making these proteins promising targets for drug development. In most cases, the catalytic domain is targeted; however, it is also possible to target membrane associations in order to regulate protein activity. In this study, we established a novel method to study protein-lipid interactions and screened for flavonoid-derived antagonists of PtdIns(3,4,5)P3 binding with the phosphoinositide-dependent kinase 1 (PDK1) pleckstrin homology (PH) domain. Using an enhanced green fluorescent protein (eGFP)-tagged PDK1 PH domain and 50% sucrose-loaded liposomes, the protein-lipid interaction could be efficiently evaluated using liposome pull-down assays coupled with fluorescence spectrophotometry, and a total of 32 flavonoids were screened as antagonists for PtdIns(3,4,5)P3 binding with the PDK1 PH domain. From this analysis, we found that two adjunct hydroxyl groups in the C ring were responsible for the inhibitory effects of the flavonoids. Because the flavonoids shared structural similarities, the results were then subjected to quantitative structure-activity relationship (QSAR) analysis. The results were then further confirmed by in silico docking experiments. Taken together, our strategy presented herein to screen antagonists targeting lipid-protein interactions could be an alternative method for identification and characterization of drug candidates.