Publications by authors named "Stephen Bergmeier"

47 Publications

A novel GSK-3 inhibitor binds to GSK-3β via a reversible, time and Cys-199-dependent mechanism.

Bioorg Med Chem 2021 Apr 27;40:116179. Epub 2021 Apr 27.

Department of Chemical and Biomolecular Engineering, Ohio University, Athens, OH 45701, United States; Biomedical Engineering Program, Ohio University, Athens, OH 45701, United States. Electronic address:

Glycogen synthase kinase-3 (GSK-3) has been implicated in numerous pathologies making GSK-3 an attractive therapeutic target. Our group has identified a compound termed COB-187 that is a potent and selective inhibitor of GSK-3. In this study, we probed the mechanism by which COB-187 inhibits GSK-3β. Progress curves, generated via real-time monitoring of kinase activity, indicated that COB-187 inhibition of GSK-3β is time-dependent and subsequent jump dilution assays revealed that COB-187 binding to GSK-3β is reversible. Further, a plot of the kinetic constant (k) versus COB-187 concentration suggested that, within the range of concentrations studied, COB-187 binds to GSK-3β via an induced-fit mechanism. There is a critical cysteine residue at the entry to the active site of GSK-3β (Cys-199). We generated a mutant version of GSK-3β wherein Cys-199 was substituted with an alanine. This mutation caused a dramatic decrease in the activity of COB-187; specifically, an IC in the nM range for wild type versus >100 µM for the mutant. A screen of COB-187 against 34 kinases that contain a conserved cysteine in their active site revealed that COB-187 is highly selective for GSK-3 indicating that COB-187's inhibition of GSK-3β via Cys-199 is specific. Combined, these findings suggest that COB-187 inhibits GSK-3β via a specific, reversible, time and Cys-199-dependent mechanism.
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http://dx.doi.org/10.1016/j.bmc.2021.116179DOI Listing
April 2021

A small-molecule pan-class I glucose transporter inhibitor reduces cancer cell proliferation in vitro and tumor growth in vivo by targeting glucose-based metabolism.

Cancer Metab 2021 Mar 26;9(1):14. Epub 2021 Mar 26.

Department of Biological Sciences, Ohio University, Athens, OH, 45701, USA.

Background: Cancer cells drastically increase the uptake of glucose and glucose metabolism by overexpressing class I glucose transporters (GLUT1-4) to meet their energy and biomass synthesis needs and are very sensitive and vulnerable to glucose deprivation. Although targeting glucose uptake via GLUTs has been an attractive anticancer strategy, the relative anticancer efficacy of multi-GLUT targeting or single GLUT targeting is unclear. Here, we report DRB18, a synthetic small molecule, is a potent anticancer compound whose pan-class I GLUT inhibition is superior to single GLUT targeting.

Methods: Glucose uptake and MTT/resazurin assays were used to measure DRB18's inhibitory activities of glucose transport and cell viability/proliferation in human lung cancer and other cancer cell lines. Four HEK293 cell lines expressing GLUT1-4 individually were used to determine the IC values of DRB18's inhibitory activity of glucose transport. Docking studies were performed to investigate the potential direct interaction of DRB18 with GLUT1-4. Metabolomics analysis was performed to identify metabolite changes in A549 lung cancer cells treated with DRB18. DRB18 was used to treat A549 tumor-bearing nude mice. The GLUT1 gene was knocked out to determine how the KO of the gene affected tumor growth.

Results: DRB18 reduced glucose uptake mediated via each of GLUT1-4 with different ICs, which match with the docking glidescores with a correlation coefficient of 0.858. Metabolomics analysis revealed that DRB18 altered energy-related metabolism in A549 cells by changing the abundance of metabolites in glucose-related pathways in vitro and in vivo. DRB18 eventually led to G1/S phase arrest and increased oxidative stress and necrotic cell death. IP injection of DRB18 in A549 tumor-bearing nude mice at 10 mg/kg body weight thrice a week led to a significant reduction in the tumor volume compared with mock-treated tumors. In contrast, the knockout of the GLUT1 gene did not reduce tumor volume.

Conclusions: DRB18 is a potent pan-class I GLUT inhibitor in vitro and in vivo in cancer cells. Mechanistically, it is likely to bind the outward open conformation of GLUT1-4, reducing tumor growth through inhibiting GLUT1-4-mediated glucose transport and metabolisms. Pan-class I GLUT inhibition is a better strategy than single GLUT targeting for inhibiting tumor growth.
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http://dx.doi.org/10.1186/s40170-021-00248-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8004435PMC
March 2021

RNA drug discovery: Conformational restriction enhances specific modulation of the T-box riboswitch function.

Bioorg Med Chem 2020 10 6;28(20):115696. Epub 2020 Aug 6.

Department of Chemistry & Biochemistry, Clippinger Laboratory, Ohio University, Athens, OH 45701, USA; Molecular & Cellular Biology Program, Ohio University, Athens, OH 45701, USA. Electronic address:

Antibacterial drug resistance is a global health concern that requires multiple solution approaches including development of new antibacterial compounds acting at novel targets. Targeting regulatory RNA is an emerging area of drug discovery. The T-box riboswitch is a regulatory RNA mechanism that controls gene expression in Gram-positive bacteria and is an exceptional, novel target for antibacterial drug design. We report the design, synthesis and activity of a series of conformationally restricted oxazolidinone-triazole compounds targeting the highly conserved antiterminator RNA element of the T-box riboswitch. Computational binding energies correlated with experimentally-derived K values indicating the predictive capabilities for docking studies within this series of compounds. The conformationally restricted compounds specifically inhibited T-box riboswitch function and not overall transcription. Complex disruption, computational docking and RNA binding specificity data indicate that inhibition may result from ligand binding to an allosteric site. These results highlight the importance of both ligand affinity and RNA conformational outcome for targeted RNA drug design.
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http://dx.doi.org/10.1016/j.bmc.2020.115696DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7716477PMC
October 2020

Isosteres of ester derived glucose uptake inhibitors.

Bioorg Med Chem Lett 2020 09 15;30(18):127406. Epub 2020 Jul 15.

Department of Chemistry and Biochemistry, Ohio University, Athens, OH 45701, USA; Program of Molecular and Cellular Biology, Ohio University, Athens, OH 45701, USA. Electronic address:

Glucose transporters (GLUTs) facilitate glucose uptake and are overexpressed in most cancer cells. Inhibition of glucose transport has been shown to be an effective method to slow the growth of cancer cells both in vitro and in vivo. We have previously reported on the anticancer activity of an ester derived glucose uptake inhibitor. Due to the hydrolytic instability of the ester linkage we have prepared a series of isosteres of the ester moiety. Of all of the isosteres prepared, the amine linkage showed the most promise. Several additional analogues of the amine-linked compounds were also prepared to improve the overall activity.
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http://dx.doi.org/10.1016/j.bmcl.2020.127406DOI Listing
September 2020

Modulation of LPS-induced inflammatory cytokine production by a novel glycogen synthase kinase-3 inhibitor.

Eur J Pharmacol 2020 Sep 4;883:173340. Epub 2020 Jul 4.

Department of Chemical and Biomolecular Engineering, Ohio University, Athens, OH, 45701, USA; Biomedical Engineering Program, Ohio University, Athens, OH, 45701, USA. Electronic address:

Sepsis is a serious condition that can lead to long-term organ damage and death. At the molecular level, the hallmark of sepsis is the elevated expression of a multitude of potent cytokines, i.e. a cytokine storm. For sepsis involving gram-negative bacteria, macrophages recognize lipopolysaccharide (LPS) shed from the bacteria, activating Toll-like-receptor 4 (TLR4), and triggering a cytokine storm. Glycogen synthase kinase-3 (GSK-3) is a highly active kinase that has been implicated in LPS-induced cytokine production. Thus, compounds that inhibit GSK-3 could be potential therapeutics for sepsis. Our group has recently described a novel and highly selective inhibitor of GSK-3 termed COB-187. In the present study, using THP-1 macrophages, we evaluated the ability of COB-187 to attenuate LPS-induced cytokine production. We found that COB-187 significantly reduced, at the protein and mRNA levels, cytokines induced by LPS (e.g. IL-6, TNF-α, IL-1β, CXCL10, and IFN-β). Further, the data suggest that the inhibition could be due, at least in part, to COB-187 reducing NF-κB (p65/p50) DNA binding activity as well as reducing IRF-3 phosphorylation at Serine 396. Thus, COB-187 appears to be a potent inhibitor of the cytokine storm induced by LPS.
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http://dx.doi.org/10.1016/j.ejphar.2020.173340DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7334664PMC
September 2020

Identification of a novel selective and potent inhibitor of glycogen synthase kinase-3.

Am J Physiol Cell Physiol 2019 12 25;317(6):C1289-C1303. Epub 2019 Sep 25.

Department of Chemical and Biomolecular Engineering, Ohio University, Athens, Ohio.

Glycogen synthase kinase-3 (GSK-3) is a multitasking protein kinase that regulates numerous critical cellular functions. Not surprisingly, elevated GSK-3 activity has been implicated in a host of diseases including pathological inflammation, diabetes, cancer, arthritis, asthma, bipolar disorder, and Alzheimer's. Therefore, reagents that inhibit GSK-3 activity provide a means to investigate the role of GSK-3 in cellular physiology and pathophysiology and could become valuable therapeutics. Finding a potent inhibitor of GSK-3 that can selectively target this kinase, among over 500 protein kinases in the human genome, is a significant challenge. Thus there remains a critical need for the identification of selective inhibitors of GSK-3. In this work, we introduce a novel small organic compound, namely COB-187, which exhibits potent and highly selective inhibition of GSK-3. Specifically, this study ) utilized a molecular screen of 414 kinase assays, representing 404 unique kinases, to reveal that COB-187 is a highly potent and selective inhibitor of GSK-3; ) utilized a cellular assay to reveal that COB-187 decreases the phosphorylation of canonical GSK-3 substrates indicating that COB-187 inhibits cellular GSK-3 activity; and ) reveals that a close isomer of COB-187 is also a selective and potent inhibitor of GSK-3. Taken together, these results demonstrate that we have discovered a region of chemical design space that contains novel GSK-3 inhibitors. These inhibitors will help to elucidate the intricate function of GSK-3 and can serve as a starting point for the development of potential therapeutics for diseases that involve aberrant GSK-3 activity.
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http://dx.doi.org/10.1152/ajpcell.00061.2019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6962522PMC
December 2019

Antitumor Activities of the Novel Isosteviol Derivative 10C Against Liver Cancer.

Anticancer Res 2017 04;37(4):1591-1601

Department of Chemistry and Biochemistry, Ohio University, Athens, OH, U.S.A.

Background/aim: Hepatocellular carcinoma (HCC) is the most common type of liver cancer and the fifth most common primary malignancy with worldwide increasing incidence. The current study aimed to investigate the anticancer activities of novel isosteviol derivatives towards human HepG2 hepatocellular cancer cells and in an animal model of hepatocellular carcinoma.

Materials And Methods: Twelve isosteviol derivatives were screened for their anti-proliferative activities against HepG2 and IC was calculated for all designed derivatives. The impact of the potent isosteviol derivative 10C on HepG2 cells was further studied by MTT assay, Annexin V/PI staining, flow cytometry and western blotting. In vivo studies were performed to assess the anticancer effect of isosteviol derivative 10C on Diethyl Nitrosamine-induced liver cancer in female rats by evaluating the physiological processes.

Results: isosteviol derivative 10C induced growth inhibition with IC of 2 μM mainly through induction of apoptosis in HepG2 cells. Additionally, isosteviol derivative 10C induced G phase arrest, which was further confirmed by increased expression of cyclin dependent kinase inhibitor 1A (CDKN1A, p21). It also increased BAX, BID and PARP-1 and while it reduced pro-CASPASE-3 expression and phosphorylation levels of AKT in HepG2 cells. Furthermore, western blotting data showed that E-cadherin, β-catenin, VEGF and COX-2 expressions were suppressed by isosteviol derivative 10C in HepG2 cells. The in vivo study demonstrated that dose-dependent treatment of isosteviol derivative 10C led to significant reduction in tumor size compared to the untreated group after the fourth injection with no significant effects on major physiological processes.

Conclusion: Taken together, in vitro and in vivo studies revealed that isosteviol derivative 10C induced apoptosis in HepG2 cells, blocked angiogenic signaling and it did not induce any apparent toxicity towards the treated hosts which merits further investigation at clinical level.
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http://dx.doi.org/10.21873/anticanres.11489DOI Listing
April 2017

Phenylmethimazole and a thiazole derivative of phenylmethimazole inhibit IL-6 expression by triple negative breast cancer cells.

Eur J Pharmacol 2017 May 23;803:130-137. Epub 2017 Mar 23.

Department of Chemical and Biomolecular Engineering, Ohio University, Athens, OH 45701, USA; Biomedical Engineering Program, Ohio University, Athens, OH 45701, USA.

Inhibition of interleukin-6 (IL-6) holds significant promise as a therapeutic approach for triple negative breast cancer (TNBC). We previously reported that phenylmethimazole (C10) reduces IL-6 expression in several cancer cell lines. We have identified a more potent derivative of C10 termed COB-141. In the present work, we tested the hypothesis that C10 and COB-141 inhibit TNBC cell expressed IL-6 and investigated the potential for classical IL-6 pathway induced signaling within TNBC cells. A panel of TNBC cell lines (MDA-MB-231, Hs578T, MDA-MB-468) was used. Enzyme linked immunosorbent assays (ELISA) revealed that C10 and COB-141 inhibit MDA-MB-231 cell IL-6 secretion, with COB-141 being ~6.5 times more potent than C10. Therefore, the remainder of the study focused on COB-141 which inhibited IL-6 secretion, and was found, via quantitative real time polymerase chain reaction (QRT-PCR), to inhibit IL-6 mRNA in the TNBC panel. COB-141 had little, if any, effect on metabolic activity indicating that the IL-6 inhibition is not via a toxic effect. Flow cytometric analysis and QRT-PCR revealed that the TNBC cell lines do not express the IL-6 receptor (IL-6Rα). Trans-AM assays suggested that COB-141 exerts its inhibitory effect, at least in part, by reducing NF-κB (p65/p50) DNA binding. In summary, COB-141 is a potent inhibitor of TNBC cell expressed IL-6 and the inhibition does not appear to be due to non-specific toxicity. The TNBC cell lines do not have an intact classical IL-6 signaling pathway. COB-141's inhibitory effect may be due, at least in part, to reducing NF-κB (p65/p50) DNA binding.
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http://dx.doi.org/10.1016/j.ejphar.2017.03.049DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5461878PMC
May 2017

Simple modifications to methimazole that enhance its inhibitory effect on tumor necrosis factor-α-induced vascular cell adhesion molecule-1 expression by human endothelial cells.

Eur J Pharmacol 2015 Mar 29;751:59-66. Epub 2015 Jan 29.

Biomedical Engineering Program, Ohio University, Athens, OH 45701, USA; Department of Chemical and Biomolecular Engineering, Ohio University, Athens, OH 45701, USA.

The expression of vascular cell adhesion molecule-1 (VCAM-1) on the vascular endothelium can be increased by pro-inflammatory cytokines [e.g. tumor necrosis factor-α (TNF-α)]. VCAM-1 contributes to leukocyte adhesion to, and emigration from, the vasculature which is a key aspect of pathological inflammation. As such, a promising therapeutic approach for pathological inflammation is to inhibit the expression of VCAM-1. Methimazole [3-methyl-1, 3 imidazole-2 thione (MMI)] is routinely used for the treatment of Graves׳ disease and patients treated with MMI have decreased levels of circulating VCAM-1. In this study we used cultured human umbilical vein endothelial cells (HUVEC) to investigate the effect of MMI structural modifications on TNF-α induced VCAM-1 expression. We found that addition of a phenyl ring at the 4-nitrogen of MMI yields a compound that is significantly more potent than MMI at inhibiting 24h TNF-α-induced VCAM-1 protein expression. Addition of a para methoxy to the appended phenyl group increases the inhibition while substitution of a thiazole ring for an imidazole ring in the phenyl derivatives yields no clear difference in inhibition. Addition of the phenyl ring to MMI appears to increase toxicity as does substitution of a thiazole ring for an imidazole ring in the phenyl MMI derivatives. Each of the compounds reduced TNF-α-induced VCAM-1 mRNA expression and had a functional inhibitory effect, i.e. each inhibited monocytic cell adhesion to 24h TNF-α-activated HUVEC under fluid flow conditions. Combined, these studies provide important insights into the design of MMI-related anti-inflammatory compounds.
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http://dx.doi.org/10.1016/j.ejphar.2015.01.032DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5019189PMC
March 2015

Synthesis and cytotoxic activity of MOM-ether analogs of isosteviol.

Bioorg Med Chem Lett 2014 Feb 6;24(4):1184-7. Epub 2014 Jan 6.

Department of Chemistry & Biochemistry, Edison Biotechnology Institute, Ohio University, Athens, OH 45701, USA. Electronic address:

Lung cancer is one of the most common malignancies worldwide. In this Letter, novel MOM-ether analogs of isosteviol were designed and synthesized to be tested for anticancer activities against H1299 lung cancer cell lines. The effects of these derivatives were studied in H1299 human large cell lung carcinoma cells that are null for p53 and compared to normal counterparts NL-20 normal lung epithelial cells. The initial screening of twelve MOM-ether analogs of isosteviol derivatives on H1299 lung cancer cells by MTT assay revealed that two derivatives (an ester and a carbamate) were the most potent in reducing cell viability. The IC50 values for these derivatives were determined to be 14 and 21 μM respectively. We compared the cytotoxicity of the best derivatives in H1299 lung cancer cells and NL-20 normal lung epithelial cells. Both derivatives showed lower cytotoxic effects on NL-20 normal lung epithelial cells. Moreover, both derivatives induced apoptosis in H1299 lung cancer cells more than NL-20 normal lung epithelial cells.
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http://dx.doi.org/10.1016/j.bmcl.2013.12.103DOI Listing
February 2014

Defining the putative inhibitory site for a selective negative allosteric modulator of human α4β2 neuronal nicotinic receptors.

ACS Chem Neurosci 2012 Sep 25;3(9):682-92. Epub 2012 May 25.

Division of Biology, California Institute of Technology, Pasadena, California 91125, USA.

Neuronal nicotinic receptors (nAChRs) have been implicated in several diseases and disorders such as autism spectrum disorders, Alzheimer's disease, Parkinson's disease, epilepsy, and nicotine addiction. To understand the role of nAChRs in these conditions, it would be beneficial to have selective molecules that target specific nAChRs in vitro and in vivo. Our laboratory has previously identified a novel allosteric site on human α4β2 nAChRs using a series of computational and in vitro approaches. At this site, we have identified negative allosteric modulators that selectively inhibit human α4β2 nAChRs, a subtype implicated in nicotine addiction. This study characterizes the allosteric site via site-directed mutagenesis. Three amino acids (Phe118, Glu60, and Thr58) on the β2 subunit were shown to participate in the inhibitory properties of the selective antagonist KAB-18 and provided insights into its antagonism of human α4β2 nAChRs. SAR studies with KAB-18 analogues and various mutant α4β2 nAChRs also provided information concerning how different physiochemical features influence the inhibition of nAChRs through this allosteric site. Together, these studies identify the amino acids that contribute to the selective antagonism of human α4β2 nAChRs at this allosteric site. Finally, these studies define the physiochemical features of ligands that influence interaction with specific amino acids in this allosteric site.
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http://dx.doi.org/10.1021/cn300035fDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3447398PMC
September 2012

A small-molecule inhibitor of glucose transporter 1 downregulates glycolysis, induces cell-cycle arrest, and inhibits cancer cell growth in vitro and in vivo.

Mol Cancer Ther 2012 Aug 11;11(8):1672-82. Epub 2012 Jun 11.

Department of Biological Science, Ohio University, Athens, OH, USA.

The functional and therapeutic importance of the Warburg effect is increasingly recognized, and glycolysis has become a target of anticancer strategies. We recently reported the identification of a group of novel small compounds that inhibit basal glucose transport and reduce cancer cell growth by a glucose deprivation-like mechanism. We hypothesized that the compounds target Glut1 and are efficacious in vivo as anticancer agents. Here, we report that a novel representative compound WZB117 not only inhibited cell growth in cancer cell lines but also inhibited cancer growth in a nude mouse model. Daily intraperitoneal injection of WZB117 at 10 mg/kg resulted in a more than 70% reduction in the size of human lung cancer of A549 cell origin. Mechanism studies showed that WZB117 inhibited glucose transport in human red blood cells (RBC), which express Glut1 as their sole glucose transporter. Cancer cell treatment with WZB117 led to decreases in levels of Glut1 protein, intracellular ATP, and glycolytic enzymes. All these changes were followed by increase in ATP-sensing enzyme AMP-activated protein kinase (AMPK) and declines in cyclin E2 as well as phosphorylated retinoblastoma, resulting in cell-cycle arrest, senescence, and necrosis. Addition of extracellular ATP rescued compound-treated cancer cells, suggesting that the reduction of intracellular ATP plays an important role in the anticancer mechanism of the molecule. Senescence induction and the essential role of ATP were reported for the first time in Glut1 inhibitor-treated cancer cells. Thus, WZB117 is a prototype for further development of anticancer therapeutics targeting Glut1-mediated glucose transport and glucose metabolism.
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http://dx.doi.org/10.1158/1535-7163.MCT-12-0131DOI Listing
August 2012

Fused ring aziridines as a facile entry into triazole fused tricyclic and bicyclic heterocycles.

Org Biomol Chem 2012 Apr 9;10(15):3080-91. Epub 2012 Mar 9.

Clippinger Laboratory, Department of Chemistry and Biochemistry, Ohio University, Athens, OH 45701, USA.

The intramolecular dipolar cycloaddition of an azide with an alkyne has provided a useful entry into triazole fused tricyclic heterocycles containing both the triazole ring and the oxazolidin-2-one ring system. The requisite azido-alkynes have been prepared via a two-step sequence from fused ring aziridines. A series of 6-12 membered rings containing both the oxazolidinone and triazole rings have been prepared. These ring systems have been designed as conformationally restrained analogs of RNA-binding oxazolidinones.
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http://dx.doi.org/10.1039/c2ob07042aDOI Listing
April 2012

3D-QSAR and 3D-QSSR models of negative allosteric modulators facilitate the design of a novel selective antagonist of human α4β2 neuronal nicotinic acetylcholine receptors.

Bioorg Med Chem Lett 2012 Feb 20;22(4):1797-813. Epub 2011 Nov 20.

Division of Pharmacology, College of Pharmacy, The Ohio State University, Columbus, OH, USA.

Subtype selective molecules for α4β2 neuronal nicotinic acetylcholine receptors (nAChRs) have been sought as novel therapeutics for nicotine cessation. α4β2 nAChRs have been shown to be involved in mediating the addictive properties of nicotine while other subtypes (i.e., α3β4 and α7) are believed to mediate the undesired effects of potential CNS drugs. To obtain selective molecules, it is important to understand the physiochemical features of ligands that affect selectivity and potency on nAChR subtypes. Here we present novel QSAR/QSSR models for negative allosteric modulators of human α4β2 nAChRs and human α3β4 nAChRs. These models support previous homology model and site-directed mutagenesis studies that suggest a novel mechanism of antagonism. Additionally, information from the models presented in this work was used to synthesize novel molecules; which subsequently led to the discovery of a new selective antagonist of human α4β2 nAChRs.
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http://dx.doi.org/10.1016/j.bmcl.2011.11.051DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3274641PMC
February 2012

Ligand-induced changes in T box antiterminator RNA stability.

Chem Biol Drug Des 2012 Feb 22;79(2):202-8. Epub 2011 Dec 22.

Department of Chemistry and Biochemistry, Ohio University, Athens, OH 45701, USA.

The T box antiterminator RNA element is an important component of the T box riboswitch that controls the transcription of vital genes in many Gram-positive bacteria. A series of 1,4-disubstituted 1,2,3-triazoles was screened in a fluorescence-monitored thermal denaturation assay to identify ligands that altered the stability of antiterminator model RNA. Several ligands were identified that significantly increased or decreased the melting temperature (T(m) ) of the RNA. The results indicate that this series of triazole ligands can alter the stability of antiterminator model RNA in a structure-dependent manner.
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http://dx.doi.org/10.1111/j.1747-0285.2011.01274.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3466076PMC
February 2012

Impact of fermentation, drying, roasting and Dutch processing on flavan-3-ol stereochemistry in cacao beans and cocoa ingredients.

Chem Cent J 2011 Sep 14;5:53. Epub 2011 Sep 14.

The Hershey Center for Health and Nutrition, The Hershey Company, 1025 Reese Avenue, Hershey, PA 17033-0805, USA.

This paper reports a systematic study of the level of flavan-3-ol monomers during typical processing steps as cacao beans are dried, fermented and roasted and the results of Dutch-processing. Methods have been used that resolve the stereoisomers of epicatechin and catechin. In beans harvested from unripe and ripe cacao pods, we find only (-)-epicatechin and (+)-catechin with (-)-epicatechin being by far the predominant isomer. When beans are fermented there is a large loss of both (-)-epicatechin and (+)-catechin, but also the formation of (-)-catechin. We hypothesize that the heat of fermentation may, in part, be responsible for the formation of this enantiomer. When beans are progressively roasted at conditions described as low, medium and high roast conditions, there is a progressive loss of (-)-epicatechin and (+)-catechin and an increase in (-)-catechin with the higher roast levels. When natural and Dutch-processed cacao powders are analyzed, there is progressive loss of both (-)-epicatechin and (+)-catechin with lesser losses of (-)-catechin. We thus observe that in even lightly Dutch-processed powder, the level of (-)-catechin exceeds the level of (-)-epicatechin. The results indicate that much of the increase in the level of (-)-catechin observed during various processing steps may be the result of heat-related epimerization from (-)-epicatechin. These results are discussed with reference to the reported preferred order of absorption of (-)-epicatechin > (+)-catechin > (-)-catechin. These results are also discussed with respect to the balance that must be struck between the beneficial impact of fermentation and roasting on chocolate flavor and the healthful benefits of chocolate and cocoa powder that result in part from the flavan-3-ol monomers.
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http://dx.doi.org/10.1186/1752-153X-5-53DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3215931PMC
September 2011

Synthesis and stereospecificity of 4,5-disubstituted oxazolidinone ligands binding to T-box riboswitch RNA.

J Med Chem 2011 Oct 31;54(19):6786-95. Epub 2011 Aug 31.

Department of Chemistry and Biochemistry, Ohio University, Athens, Ohio 45701, United States.

The enantiomers and the cis isomers of two previously studied 4,5-disubstituted oxazolidinones have been synthesized, and their binding to the T-box riboswitch antiterminator model RNA has been investigated in detail. Characterization of ligand affinities and binding site localization indicates that there is little stereospecific discrimination for binding antiterminator RNA alone. This binding similarity between enantiomers is likely due to surface binding, which accommodates ligand conformations that result in comparable ligand-antiterminator contacts. These results have significant implications for T-box antiterminator-targeted drug discovery and, in general, for targeting other medicinally relevant RNA that do not present deep binding pockets.
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http://dx.doi.org/10.1021/jm2006904DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3466060PMC
October 2011

Synthesis of a functionalized oxabicyclo[2.2.1]-heptene-based chemical library.

Comb Chem High Throughput Screen 2012 Jan;15(1):81-9

Department of Chemistry & Biochemistry, Ohio University, Athens, Ohio 45701, USA.

The 7-oxabicyclo[2.2.1]heptene ring system is a common structural motif in many pharmacologically interesting molecules. We recognized the potential to employ this highly oxygenated and conformationally-restricted scaffold in diversity-oriented synthesis to generate a library of non-chiral but topologically complex compounds. Herein, we report the synthesis and biological evaluation of two 96-member tricyclic libraries containing the oxabicyclo[2.2.1]heptene framework using acetal formation as the key step.
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http://dx.doi.org/10.2174/138620712798280835DOI Listing
January 2012

Structure-activity studies of RNA-binding oxazolidinone derivatives.

Bioorg Med Chem Lett 2011 Aug 15;21(15):4524-7. Epub 2011 Jun 15.

Department of Chemistry and Biochemistry, Ohio University, Athens, OH 45701, USA.

The structure-activity relationship of a series of oxazolidinones binding to T-box riboswitch antiterminator RNA has been investigated. Oxazolidinones differentially substituted at C-5 were prepared and the ligand-induced fluorescence resonance energy transfer (FRET) changes in FRET-labeled antiterminator model RNA were assayed. Both qualitative and quantitative analysis of the structure-activity relationship indicate that hydrogen bonding and hydrophobic properties play a significant role in ligand binding.
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http://dx.doi.org/10.1016/j.bmcl.2011.05.130DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3466080PMC
August 2011

Differential apoptotic effects of novel quinuclidinone analogs 8a and 8b in normal and lung cancer cell lines.

Anticancer Res 2011 Apr;31(4):1345-57

Biochemistry Department, Alexandria University, Moharam, Beck, Alexandria, Egypt.

Background: We previously reported novel quinuclidinone analogs that showed both additive and synergistic cytotoxicity in lung cancer cells. We aimed at understanding the mechanism of these analogs and also their cytotoxic effect on normal cells. The effects of these analogs were studied in response to gamma radiation in H1299 human large cell lung carcinoma cells that are null for p53, normal lung epithelial cell line (NL-20) and H1299 cells stably transfected with p53.

Materials And Methods: The effects of the analogs were investigated by MTT assay, clonogenic survival assay, sphingomylinase activity, Cox-2 activity, ELISA-based apoptotic assay, terminal deoxynucleotidyl transferase dUTP nick end labeling assay, immunofluoresence staining, flow cytometry, real-time reverse transcription polymerase chain reaction and Western blot analysis.

Results: Our data indicated that 8a and 8b reduced cell proliferation and induced apoptosis in H1299 cells more than H1299-wt p53 cells and NL-20 cells, they also radiosensitize H1299 cells to gamma radiation more than NL-20 cells. 8a and 8b decreased cells in G(2) phase in H1299 cells more than NL-20 cells, which is confirmed by increased expression of cyclin B in H1299 cells, with no significant increase in H1299-wtp53. 8a increased sphingomylinase activity and ceramide level in H1299 more than the rest of cells, it also reduced expression level and activity of COX-2 while it increased caspase-3 activity and induced PARP-1 cleavage. Both derivatives increased expression of p53 in H1299-wt p53 level, while they did not show significant increase in NL-20 cells. Interestingly, these analogs induced apoptosis in H1299 and p53 stably transfected H1299 cells, but they had less effect on normal lung epithelial cells (NL-20).

Conclusion: All these results confirm that our quinuclidinone derivatives provoke cytotoxicity in lung cancer cells more than normal cells, which is a feature not present in most chemotherapeutic drugs.
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April 2011

Novel quinuclidinone derivative 8a induced apoptosis in human MCF-7 breast cancer cell lines.

Anticancer Res 2011 Mar;31(3):871-80

Biochemistry Department, Faculty of Science, Alexandria University, Alexandria, Egypt.

Novel quinuclidinone derivatives that cause cytotoxicity in human non-small lung carcinoma epithelial cells null for p53 (H1299) have been previously reported. The current study investigates the effect of these derivatives on cytotoxicity of human MCF-7 cells and normal breast epithelial cells (MCF-12a). This study shows that quinuclidinone derivatives 8a and 8b induce growth inhibition mainly through apoptosis of breast cancer cells (MCF-7) with less cytotoxic effect in normal breast epithelial cells (MCF-12a) for derivative 8a while 8b induced similar cytotoxicity for both breast cancer cells and normal breast epithelial cells. Derivative 8a was chosen for further investigation. 8a induced G(1) phase arrest, presumably sensitizing the breast cancer cells to apoptosis by increasing expression level of p21 and cyclin E. Moreover, 8a increased expression level of ERK1, p53 and BAX, and it reduced expression level of AKT and BCL-2. By investigating the sphingomyelinase apoptosis pathway, it was observed that 8a significantly increased sphingomyelinase activity and increased formation of ceramide as well as increased expression levels of JNK phoshorylation, caspase-8 and caspase-9. Based on previous results it is proposed that quinuclidinone derivative 8a provokes apoptosis in human breast cancer cells (MCF-7) via the sphingomyelinase pathway.
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March 2011

Natural product derivatives with bactericidal activity against Gram-positive pathogens including methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus faecalis.

Bioorg Med Chem Lett 2010 Oct 7;20(19):5936-8. Epub 2010 Jul 7.

Department of Chemistry, University of Montana, Missoula, MT 59812, USA.

We have shown that the intentional engineering of a natural product biosynthesis pathway is a useful way to generate stereochemically complex scaffolds for use in the generation of combinatorial libraries that capture the structural features of both natural products and synthetic compounds. Analysis of a prototype library based upon nonactic acid lead to the discovery of triazole-containing nonactic acid analogs, a new structural class of antibiotic that exhibits bactericidal activity against drug resistant, Gram-positive pathogens including Staphylococcus aureus and Enterococcus faecalis.
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http://dx.doi.org/10.1016/j.bmcl.2010.06.146DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5443665PMC
October 2010

Small compound inhibitors of basal glucose transport inhibit cell proliferation and induce apoptosis in cancer cells via glucose-deprivation-like mechanisms.

Cancer Lett 2010 Dec 1;298(2):176-85. Epub 2010 Aug 1.

Department of Biological Science, Ohio University, Athens, OH 45701, USA.

Cancer cells depend heavily on glucose as both energy and biosynthesis sources and are found to upregulate glucose transport and switch their main energy supply pathway from oxidative phosphorylation to glycolysis. These molecular and metabolic changes also provide targets for cancer treatment. Here we report that novel small molecules inhibited basal glucose transport and cell proliferation, and induced apoptosis in lung and breast cancer cells without affecting much their normal cell counterparts. Cancer cells survived the compound treatment lost their capability to proliferate. Mechanistic study indicates that the cancer cell inhibition by the test compounds has a component of apoptosis and the induced apoptosis was p53-independent and caspase 3-dependent, similar to those resulted from glucose deprivation. Compound treatment also led to cell cycle arrest in G1/S phase. The inhibition of cancer cell growth was partially relieved when additional glucose was supplied to cells, suggesting that the inhibition was due to, at least in part, the inhibition of basal glucose transport. When used in combination, the test compounds demonstrated synergistic effects with anticancer drugs cisplatin or paclitaxel in inhibition of cancer cell growth. All these results suggest that these glucose transport inhibitors mimic glucose deprivation and work through inhibiting basal glucose transport. These inhibitors have the potential to complement and replace traditional glucose deprivation, which cannot be used in animals, as new tools to study the effects of glucose transport and metabolism on cancer and normal cells.
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http://dx.doi.org/10.1016/j.canlet.2010.07.002DOI Listing
December 2010

Library of 1,4-disubstituted 1,2,3-triazole analogs of oxazolidinone RNA-binding agents.

J Comb Chem 2010 Jul;12(4):491-6

Department of Chemistry and Biochemistry, Ohio University, Athens, Ohio 45701, USA.

The design and synthesis of small molecules that target RNA is immensely important in antibacterial therapy. We had previously reported on the RNA binding of a series of 4,5-disubstituted 2-oxazolidinones that bind to a highly conserved bulge region of bacterial RNA. This biological target T box antitermination system, which is found mainly in Gram-positive bacteria, regulates the expression of several amino acid related genes. In an effort to amplify our library, we have prepared a library of 1,4-disubstituted 1,2,3-triazole analogs that entails an isosteric replacement of the oxazolidinone nucleus. The synthesis of the new analogs was enhanced via copper(I) catalysis of an azide and alkyne cycloaddition reaction. A total of 108 1,4-disubstituted 1,2,3-triazole compounds have been prepared. All compounds were evaluated as RNA binding agents.
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http://dx.doi.org/10.1021/cc100029yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2925680PMC
July 2010

Negative allosteric modulators that target human alpha4beta2 neuronal nicotinic receptors.

J Pharmacol Exp Ther 2010 Sep 15;334(3):761-74. Epub 2010 Jun 15.

Division of Pharmacology, College of Pharmacy, Ohio State University, Columbus, Ohio 43210, USA.

Allosteric modulation of neuronal nicotinic acetylcholine receptors (nAChRs) is considered to be one of the most promising approaches for therapeutics. We have previously reported on the pharmacological activity of several compounds that act as negative allosteric modulators (NAMs) of nAChRs. In the following studies, the effects of 30 NAMs from our small chemical library on both human alpha4beta2 (Halpha4beta2) and human alpha3beta4 (Halpha3beta4) nAChRs expressed in human embryonic kidney ts201 cells were investigated. During calcium accumulation assays, these NAMs inhibited nAChR activation with IC(50) values ranging from 2.4 microM to more than 100 microM. Several NAMs showed relative selectivity for Halpha4beta2 nAChRs with IC(50) values in the low micromolar range. A lead molecule, KAB-18, was identified that shows relative selectivity for Halpha4beta2 nAChRs. This molecule contains three phenyl rings, one piperidine ring, and one ester bond linkage. Structure-activity relationship (SAR) analyses of our data revealed three regions of KAB-18 that contribute to its relative selectivity. Predictive three-dimensional quantitative SAR (comparative molecular field analysis and comparative molecular similarity indices analysis) models were generated from these data, and a pharmacophore model was constructed to determine the chemical features that are important for biological activity. Using docking approaches and molecular dynamics on a Halpha4beta2 nAChR homology model, a binding mode for KAB-18 at the alpha/beta subunit interface that corresponds to the predicted pharmacophore is described. This binding mode was supported by mutagenesis studies. In summary, these studies highlight the importance of SAR, computational, and molecular biology approaches for the design and synthesis of potent and selective antagonists targeting specific nAChR subtypes.
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http://dx.doi.org/10.1124/jpet.110.168211DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2939664PMC
September 2010

Novel inhibitors of basal glucose transport as potential anticancer agents.

Bioorg Med Chem Lett 2010 Apr 11;20(7):2191-4. Epub 2010 Feb 11.

Department of Chemistry & Biochemistry, Ohio University, Athens, OH 45701, USA.

Cancer cells commonly show increased levels of glucose uptake and dependence. A potential strategy for the treatment of cancer may be the inhibition of basal glucose transport. We report here the synthesis of a small library of polyphenolic esters that inhibit basal glucose transport in H1299 lung and other cancer cells. These basal glucose transport inhibitors also inhibit cancer cell growth in H1299 cells, and these two activities appear to be correlated.
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http://dx.doi.org/10.1016/j.bmcl.2010.02.027DOI Listing
April 2010

Ring expansion of substituted norbornadienes for the synthesis of mono- and disubstituted 2-azabicyclo[3.2.1]octadienes.

Tetrahedron Lett 2008 Sep;49(37):5363-5365

Department of Chemistry & Biochemistry, Clippinger Laboratories, Ohio University, Athens, Ohio, 45701, USA.

We have studied the conversion of substituted norbornadienes into a substituted 2-azabicyclo[3.2.1]octadiene system via reaction with toluenesulfonyl azide. We have found that both and mono- and disubstituted norbornadienes will undergo the cycloaddition/rearrangement sequence to provide the bicyclooctadiene ring system as a single regioisomer. The 2-azabicyclo[3.2.1]octane ring system can be prepared from the unsaturated 2-azabicyclo[3.2.1]octadiene ring system.
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http://dx.doi.org/10.1016/j.tetlet.2008.06.123DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2597873PMC
September 2008

Diastereoselective Synthesis of a Highly Substituted cis-Decahydroquinoline via a Knoevenagel Condensation.

Tetrahedron 2008 Jun;64(27):6434-6439

Department of Chemistry & Biochemistry, Ohio University, Athens, Ohio, 45701, USA.

A diastereoselective approach to 3,7,8-trisubstituted cis-decahydroquinolines is described. This ring system forms the core of rings B and E of the norditerpenoid alkaloid methyllycaconitine. This approach starts with a known disubsituted cyclohexene. The remaining carbons are attached via a Knoevenagel condensation followed by an intramolecular lactam formation. The stereochemistry of the substituents is controlled by the cis-substitution of the starting cyclohexene ring.
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http://dx.doi.org/10.1016/j.tet.2008.04.073DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2597876PMC
June 2008

Effect of novel negative allosteric modulators of neuronal nicotinic receptors on cells expressing native and recombinant nicotinic receptors: implications for drug discovery.

J Pharmacol Exp Ther 2009 Feb 4;328(2):504-15. Epub 2008 Nov 4.

Division of Pharmacology, College of Pharmacy, The Ohio State University, Columbus, OH 43210, USA.

Allosteric modulation of nAChRs is considered to be one of the most promising approaches for drug design targeting nicotinic acetylcholine receptors (nAChRs). We have reported previously on the pharmacological activity of several compounds that seem to act noncompetitively to inhibit the activation of alpha3beta4(*) nAChRs. In this study, the effects of 51 structurally similar molecules on native and recombinant alpha3beta4 nAChRs are characterized. These 51 molecules inhibited adrenal neurosecretion activated via stimulation of native alpha3beta4(*) nAChR, with IC(50) values ranging from 0.4 to 13.0 microM. Using cells expressing recombinant alpha3beta4 nAChRs, these molecules inhibited calcium accumulation (a more direct assay to establish nAChR activity), with IC(50) values ranging from 0.7 to 38.2 microM. Radiolabeled nAChR binding studies to orthosteric sites showed no inhibitory activity on either native or recombinant nAChRs. Correlation analyses of the data from both functional assays suggested additional, non-nAChR activity of the molecules. To test this hypothesis, the effects of the drugs on neurosecretion stimulated through non-nAChR mechanisms were investigated; inhibitory effects ranged from no inhibition to 95% inhibition at concentrations of 10 microM. Correlation analyses of the functional data confirmed this hypothesis. Several of the molecules (24/51) increased agonist binding to native nAChRs, supporting allosteric interactions with nAChRs. Computational modeling and blind docking identified a binding site for our negative allosteric modulators near the orthosteric binding site of the receptor. In summary, this study identified several molecules for potential development as negative allosteric modulators and documented the importance of multiple screening assays for nAChR drug discovery.
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http://dx.doi.org/10.1124/jpet.108.144576DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2682284PMC
February 2009

Natural products in parallel synthesis: triazole libraries of nonactic acid.

Bioorg Med Chem Lett 2008 Jul 10;18(14):3946-9. Epub 2008 Jun 10.

Promiliad Biopharma, 340 W. State Street Athens, OH 45701, USA.

The synthesis of a library of nonactic acid-derived triazoloamide derivatives and their evaluation as antimicrobial agents is described.
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http://dx.doi.org/10.1016/j.bmcl.2008.06.020DOI Listing
July 2008