39 results on '"Days, Emily"'
Search Results
2. A Duplexed High-Throughput Screen to Identify Allosteric Modulators of the Glucagon-Like Peptide 1 and Glucagon Receptors
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Morris, Lindsey C., Days, Emily L., Turney, Maxine, Mi, Dehui, Lindsley, Craig W., Weaver, C. David, and Niswender, Kevin D.
- Published
- 2014
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3. Identification of Potent, Selective, and Peripherally Restricted Serotonin Receptor 2B Antagonists from a High-Throughput Screen.
- Author
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Bender, Aaron M., Valentine, Michael S., Bauer, Joshua A., Days, Emily, Lindsley, Craig W., and Merryman, W. David
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SEROTONIN antagonists ,SEROTONIN receptors ,HIGH throughput screening (Drug development) ,PULMONARY arterial hypertension ,HEART valve diseases ,BLOOD-brain barrier - Abstract
Antagonists of the serotonin receptor 2B (5-HT
2B ) have shown great promise as therapeutics for the treatment of pulmonary arterial hypertension, valvular heart disease, and related cardiopathies. Herein, we describe a high-throughput screen campaign that led to the identification of highly potent and selective 5-HT2B antagonists. Furthermore, selected compounds were profiled for their predicted ability to cross the blood–brain barrier. Two exemplary compounds, VU0530244 and VU0631019, were predicted to have very limited potential for brain penetration in human subjects, a critical profile for the development of 5-HT2B antagonists devoid of centrally-mediated adverse effects. [ABSTRACT FROM AUTHOR]- Published
- 2023
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4. Small-Molecule Screen Identifies Inhibitors of the Neuronal K-Cl Cotransporter KCC2
- Author
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Delpire, Eric, Days, Emily, Lewis, L. Michelle, Mi, Dehui, Kim, Kwangho, Lindsley, Craig W., Weaver, C. David, and Jan, Lily Y.
- Published
- 2009
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5. Rosalind Franklin Society Proudly Announces the 2023 Award Recipient for ASSAY and Drug Development Technologies.
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Days, Emily Laura Krozel
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HIGH throughput screening (Drug development) ,DRUG discovery ,PULMONARY arterial hypertension ,HEART valve diseases ,SMALL molecules ,SEROTONIN receptors ,SEROTONIN antagonists ,MUSCARINIC receptors - Abstract
The Rosalind Franklin Society (RFS) has announced the recipient of the 2023 RFS Award in Science for the journal "Assay & Drug Development Technologies." The award recognizes the outstanding research and published work of women and underrepresented minority scientists, physicians, and engineers. The winning article, titled "Identification of Potent, Selective, and Peripherally Restricted Serotonin Receptor 2B Antagonists from a High-Throughput Screen," discusses the discovery of highly potent and selective 5-HT2B antagonists, which have potential therapeutic applications for pulmonary arterial hypertension and valvular heart disease. The article also highlights two compounds, VU0530244 and VU0631019, that are predicted to have limited potential for brain penetration, making them suitable for the development of 5-HT2B antagonists without centrally-mediated adverse effects. The article is authored by Aaron M. Bender, Michael S. Valentine, Joshua A. Bauer, Emily Days, Craig W. Lindsley, and W. David Merryman. The abstract provides an overview of the research findings, and the biosketch introduces Emily Days, a Data Analyst at the Vanderbilt University High-Throughput Screening Facility, who played a significant role in the awarded project. [Extracted from the article]
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- 2024
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6. Pro-inflammatory cytokines modify neuronal nicotinic acetylcholine receptor assembly
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Gahring, Lorise C., Days, Emily L., Kaasch, Tuesday, González de Mendoza, Mónica, Owen, Leah, Persiyanov, Karina, and Rogers, Scott W.
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- 2005
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7. Optimization of an ether series of mGlu5 positive allosteric modulators: Molecular determinants of MPEP-site interaction crossover
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Manka, Jason T., Vinson, Paige N., Gregory, Karen J., Zhou, Ya, Williams, Richard, Gogi, Kiran, Days, Emily, Jadhav, Satya, Herman, Elizabeth J., Lavreysen, Hilde, Mackie, Claire, Bartolomé, José M., Macdonald, Gregor J., Steckler, Thomas, Daniels, J. Scott, Weaver, C. David, Niswender, Colleen M., Jones, Carrie K., Conn, P. Jeffrey, Lindsley, Craig W., and Stauffer, Shaun R.
- Published
- 2012
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8. Discovery and optimization of a novel, selective and brain penetrant M 1 positive allosteric modulator (PAM): The development of ML169, an MLPCN probe
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Reid, Paul R., Bridges, Thomas M., Sheffler, Douglas J., Cho, Hyekyung P., Lewis, L. Michelle, Days, Emily, Daniels, J. Scott, Jones, Carrie K., Niswender, Colleen M., Weaver, C. David, Conn, P. Jeffrey, Lindsley, Craig W., and Wood, Michael R.
- Published
- 2011
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9. Screening for AMPA receptor auxiliary subunit specific modulators.
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Azumaya, Caleigh M., Days, Emily L., Vinson, Paige N., Stauffer, Shaun, Sulikowski, Gary, Weaver, C. David, and Nakagawa, Terunaga
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AMPA receptors , *NEURAL transmission , *MATERIAL plasticity , *PATHOLOGICAL psychology , *CATIONS - Abstract
AMPA receptors (AMPAR) are ligand gated ion channels critical for synaptic transmission and plasticity. Their dysfunction is implicated in a variety of psychiatric and neurological diseases ranging from major depressive disorder to amyotrophic lateral sclerosis. Attempting to potentiate or depress AMPAR activity is an inherently difficult balancing act between effective treatments and debilitating side effects. A newly explored strategy to target subsets of AMPARs in the central nervous system is to identify compounds that affect specific AMPAR-auxiliary subunit complexes. This exploits diverse spatio-temporal expression patterns of known AMPAR auxiliary subunits, providing means for designing brain region-selective compounds. Here we report a high-throughput screening-based pipeline that can identify compounds that are selective for GluA2-CNIH3 and GluA2-stargazin complexes. These compounds will help us build upon the growing library of AMPAR-auxiliary subunit specific inhibitors, which have thus far all been targeted to TARP γ-8. We used a cell-based assay combined with a voltage-sensitive dye (VSD) to identify changes in glutamate-gated cation flow across the membranes of HEK cells co-expressing GluA2 and an auxiliary subunit. We then used a calcium flux assay to further validate hits picked from the VSD assay. VU0612951 and VU0627849 are candidate compounds from the initial screen that were identified as negative and positive allosteric modulators (NAM and PAM), respectively. They both have lower IC50/EC50s on complexes containing stargazin and CNIH3 than GSG1L or the AMPAR alone. We have also identified a candidate compound, VU0539491, that has NAM activity in GluA2(R)-CNIH3 and GluA2(Q) complexes and PAM activity in GluA2(Q)-GSG1L complexes. [ABSTRACT FROM AUTHOR]
- Published
- 2017
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10. Discovery of a Novel Series of Orally Bioavailable and CNS Penetrant Glucagon-like Peptide-1 Receptor (GLP-1R) Noncompetitive Antagonists Based on a 1,3-Disubstituted-7-aryl-5,5-bis(trifluoromethyl)-5,8-dihydropyrimido[4,5-d]pyrimidine-2,4(1H,3H)-dione Core
- Author
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Nance, Kellie D., Days, Emily L., Weaver, C. David, Coldren, Anastasia, Farmer, Tiffany D., Cho, Hyekyung P., Niswender, Colleen M., Blobaum, Anna L., Niswender, Kevin D., and Lindsley, Craig W.
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GLUCAGON-like peptide-1 receptor , *URACIL , *DRUG bioavailability , *EXENDINS , *BLOOD sugar - Abstract
A duplexed, functional multiaddition high throughput screen and subsequent optimization effort identified the first orally bioavailable and CNS penetrant glucagon-like peptide-1 receptor (GLP-1R) noncompetitive antagonist. Antagonist 5d not only blocked exendin-4-stimulated insulin release in islets but also lowered insulin levels while increasing blood glucose in vivo. [ABSTRACT FROM AUTHOR]
- Published
- 2017
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11. Discovery and Characterization of a Potent and Selective Inhibitor of Aedes aegypti Inward Rectifier Potassium Channels.
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Raphemot, Rene, Rouhier, Matthew F., Swale, Daniel R., Days, Emily, Weaver, C. David, Lovell, Kimberly M., Konkel, Leah C., Engers, Darren W., Bollinger, Sean F., Hopkins, Corey, Piermarini, Peter M., and Denton, Jerod S.
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AEDES aegypti ,VIRUS disease transmission ,POTASSIUM channels ,DISEASE vectors ,INSECTICIDE resistance ,ELECTROPHYSIOLOGY - Abstract
Vector-borne diseases such as dengue fever and malaria, which are transmitted by infected female mosquitoes, affect nearly half of the world's population. The emergence of insecticide-resistant mosquito populations is reducing the effectiveness of conventional insecticides and threatening current vector control strategies, which has created an urgent need to identify new molecular targets against which novel classes of insecticides can be developed. We previously demonstrated that small molecule inhibitors of mammalian Kir channels represent promising chemicals for new mosquitocide development. In this study, high-throughput screening of approximately 30,000 chemically diverse small-molecules was employed to discover potent and selective inhibitors of Aedes aegypti Kir1 (AeKir1) channels heterologously expressed in HEK293 cells. Of 283 confirmed screening ‘hits’, the small-molecule inhibitor VU625 was selected for lead optimization and in vivo studies based on its potency and selectivity toward AeKir1, and tractability for medicinal chemistry. In patch clamp electrophysiology experiments of HEK293 cells, VU625 inhibits AeKir1 with an IC
50 value of 96.8 nM, making VU625 the most potent inhibitor of AeKir1 described to date. Furthermore, electrophysiology experiments in Xenopus oocytes revealed that VU625 is a weak inhibitor of AeKir2B. Surprisingly, injection of VU625 failed to elicit significant effects on mosquito behavior, urine excretion, or survival. However, when co-injected with probenecid, VU625 inhibited the excretory capacity of mosquitoes and was toxic, suggesting that the compound is a substrate of organic anion and/or ATP-binding cassette (ABC) transporters. The dose-toxicity relationship of VU625 (when co-injected with probenecid) is biphasic, which is consistent with the molecule inhibiting both AeKir1 and AeKir2B with different potencies. This study demonstrates proof-of-concept that potent and highly selective inhibitors of mosquito Kir channels can be developed using conventional drug discovery approaches. Furthermore, it reinforces the notion that the physical and chemical properties that determine a compound's bioavailability in vivo will be critical in determining the efficacy of Kir channel inhibitors as insecticides. [ABSTRACT FROM AUTHOR]- Published
- 2014
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12. Development and Validation of Fluorescence-Based and Automated Patch Clamp-Based Functional Assays for the Inward Rectifier Potassium Channel Kir4.1.
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Raphemot, Rene, Kadakia, Rishin J., Olsen, Michelle L., Banerjee, Sreedatta, Days, Emily, Smith, Stephen S., Weaver, C. David, and Denton, Jerod S.
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FLUORESCENCE ,PATCH-clamp techniques (Electrophysiology) ,POTASSIUM channels ,BIOLOGICAL assay ,FLUOXETINE ,TEMPORAL lobe epilepsy ,HYPERTENSION ,MOLECULAR pharmacology - Abstract
The inward rectifier potassium (Kir) channel Kir4.1 plays essential roles in modulation of neurotransmission and renal sodium transport and may represent a novel drug target for temporal lobe epilepsy and hypertension. The molecular pharmacology of Kir4.1 is limited to neurological drugs, such as fluoxetine (Prozac
© ), exhibiting weak and nonspecific activity toward the channel. The development of potent and selective small-molecule probes would provide critically needed tools for exploring the integrative physiology and therapeutic potential of Kir4.1. A fluorescence-based thallium (Tl+ ) flux assay that utilizes a tetracycline-inducible T-Rex-HEK293-Kir4.1 cell line to enable high-throughput screening (HTS) of small-molecule libraries was developed. The assay is dimethyl sulfoxide tolerant and exhibits robust screening statistics (Z′=0.75±0.06). A pilot screen of 3,655 small molecules and lipids revealed 16 Kir4.1 inhibitors (0.4% hit rate). 3,3-Diphenyl-N-(1-phenylethyl)propan-1-amine, termed VU717, inhibits Kir4.1-mediated thallium flux with an IC50 of ∼6 μM. An automated patch clamp assay using the IonFlux HT workbench was developed to facilitate compound characterization. Leak-subtracted ensemble 'loose patch' recordings revealed robust tetracycline-inducible and Kir4.1 currents that were inhibited by fluoxetine (IC50 =10 μM), VU717 (IC50 =6 μM), and structurally related calcium channel blocker prenylamine (IC50 =6 μM). Finally, we demonstrate that VU717 inhibits Kir4.1 channel activity in cultured rat astrocytes, providing proof-of-concept that the Tl+ flux and IonFlux HT assays can enable the discovery of antagonists that are active against native Kir4.1 channels. [ABSTRACT FROM AUTHOR]- Published
- 2013
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13. DihydrothiazolopyridoneDerivatives as a Novel Familyof Positive Allosteric Modulators of the Metabotropic Glutamate 5(mGlu5) Receptor.
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Bartolomé-Nebreda, José Manuel, Conde-Ceide, Susana, Delgado, Francisca, Iturrino, Laura, Pastor, Joaquín, Pena, Miguel Ángel, Trabanco, Andrés A., Tresadern, Gary, Wassvik, Carola M., Stauffer, Shaun R., Jadhav, Satyawan, Gogi, Kiran, Vinson, Paige N., Noetzel, Meredith J., Days, Emily, Weaver, C. David, Lindsley, Craig W., Niswender, Colleen M., Jones, Carrie K., and Conn, P. Jeffrey
- Published
- 2013
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14. Small-molecule screen identifies inhibitors of the neuronal K-Cl cotransporter KCC2.
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DeIpire, Eric, Days, Emily, Lewis, L. Michelle, Dehui Mi, Kwangho Kim, Lindsley, Craig W., and Weaver, C. David
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NEURAL circuitry , *GABA receptors , *DIURETICS , *THALLIUM isotopes , *FLUORESCENCE microscopy , *SODIUM/POTASSIUM ATPase , *POTASSIUM chloride - Abstract
KCC2, a neuronal-specific K-Cl cotransporter, plays a major role in maintaining intracellular Cl concentration in neurons below its electrochemical equilibrium potential, thus favoring robust GABA hyperpolarizing or inhibitory responses. The pharmacology of the K-Cl cotransporter is dominated by loop diuretics such as furosemide and bumetanide, molecules used in clinical medicine because they inhibit the loop of Henle Na-K-2Cl cotransporter with much higher affinity. To identify molecules that affect KCC2 activity, we developed a fluorescence-based assay suitable for highthroughput screening (HTS) and used the assay to screen a library of 234,000 small molecules. We identified a large number of molecules that either decrease or increase the activity of the cotransporter. Here, we report the characterization of a small number of inhibitors, some of which inhibit KCC2 activity in the submicomolar range without substantially affecting NKCC1 activity. Using medicinal chemistry, we synthesized a number of variants, tested their effect on KCC2 function, and provide an analysis of structure/activity relationships. We also used one of the compounds to demonstrate competitive inhibition in regard to external tK] versus noncompetitive inhibition in respect to external [Cl]. [ABSTRACT FROM AUTHOR]
- Published
- 2009
15. Age-related loss of neuronal nicotinic receptor expression in the aging mouse hippocampus corresponds with cyclooxygenase-2 and PPARγ expression and is altered by long-term NS398 administration.
- Author
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Gahring, Lorise C., Persiyanov, Karina, Days, Emily L., and Rogers, Scott W.
- Abstract
Age-related changes in the mammalian dorsal hippocampus are associated with diminished expression of neuronal nicotinic acetylcholine receptors (nAChR), which is particularly severe in pathologies such as those associated with dementias, including Alzheimer's disease. Because the mouse is a useful model for age-related decline in nAChR expression in the basal forebrain and limbic system, we used immunohistochemistry to examine the influence of long-term (12-month) oral administration of nicotine and/or the cyclooxygenase-2 (COX-2) preferring non-steroidal anti-inflammatory drug (NSAID) NS398 on nAChRα4, α5, α7, and β4 expression in the C57BL/6 mouse. Inhibitory neurons of the dorsal hippocampus that express nAChRs also constitutively express COX-2 and the peroxisome proliferator-antagonist receptor subtype gamma-2 (PPARγ2) which is also a target of NS398. Administration of NS398 correlated with retention of nAChRα4 and to a lesser extent nAChRβ4, but not nAChRα5 or α7, but nicotine exhibited no similar effect. Nicotine and NS398 co-administration abolished the NS398-related effect on nAChRα4 retention. These results provide evidence that the interaction during aging between oral administration of nicotine and NSAIDs are not straightforward and could even be antagonistic when combined. © 2004 Wiley Periodicals, Inc. J Neurobiol, 2005 [ABSTRACT FROM AUTHOR]
- Published
- 2005
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16. 225-LB: Targeting ß-Cell Endoplasmic Reticulum (ER) TALK-1 Channels to Restore Ca2+ Homeostasis and Reduce ER Stress in Diabetes.
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NAKHE, ARYA Y., DADI, PRASANNA, WESTOVER, DAVID, DAYS, EMILY, DENTON, JEROD, and JACOBSON, DAVID
- Abstract
Perturbations in Ca
2+ homeostasis contribute to β-cell failure in diabetes, limiting insulin secretion and exacerbating hyperglycemia. TALK-1 is the most islet-restricted and most abundantly expressed K+ channel of the β-cell. ER TALK-1 channels provide K+ countercurrent for [Ca2+ ]ER release, thereby tuning [Ca2+ ]ER storage and release. During β-cell Ca2+ influx, TALK-1-enhanced [Ca2+ ]ER release leads to activation of the hyperpolarizing Ca2+ activated K+ channels, thus increasing the silent phase duration between Ca2+ oscillations. A gain-of-function mutation in TALK-1(L114P) causes MODY, by disrupting cytoplasmic and ER Ca2+ handling. However, the ability to probe ER TALK-1 channels as a therapeutic target to normalize β-cell Ca2+ handling and reduce ER stress in diabetes has not been determined. Here, using a high throughput fluorescence-based thallium (TI+ ) flux assay (z' =0.64) in a TALK-1 overexpression cell line, we identified 80 TALK-1 selective inhibitors from a small molecule library of 50,000 compounds. 4 potent inhibitors that also inhibited the ER membrane TALK-1 channels were identified by monitoring cyclopiazonic acid-induced [Ca2+ ]ER release (e.g. K2P16-i7(IC50 =5.3μM); 63.36% [Ca2+ ]ER recovery, p-value=0.02). We also tested these inhibitors for their efficacy in controlling mouse islet [Ca2+ ]ER storage as well as cytosolic Ca2+ oscillations. TALK-1 inhibitors significantly increased islet [Ca2+ ]ER storage and Ca2+ oscillation frequency (e.g.K2P16-i7; [Ca2+ ]ER AUC=60.9a.u., peaks/min=0.45 vs. DMSO; [Ca2+ ]ER AUC=49.7a.u., peaks/min= 0.17, p-value<0.05). Importantly, the inhibitors did not alter [Ca2+ ]ER and glucose-stimulated Ca2+ oscillation frequency in TALK-1 KO islets. In summary, we have identified the first ER TALK-1 selective inhibitors that can not only be utilized to assess the role of TALK-1 in human β-cell Ca2+ handling and GSIS, but also potentially reduce ER dysfunction during diabetes pathogenesis. Disclosure: A. Y. Nakhe: None. P. Dadi: None. D. Westover: None. E. Days: None. J. Denton: None. D. Jacobson: None. [ABSTRACT FROM AUTHOR]- Published
- 2021
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17. 2075-P: Testing the First Selective Talk-1 Inhibitors for Their Ability to Improve ß-Cell Function in Diabetes.
- Author
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NAKHE, ARYA Y., DADI, PRASANNA, WESTOVER, DAVID, DAYS, EMILY, DENTON, JEROD, and JACOBSON, DAVID
- Abstract
Insulin levels are inadequate to maintain euglycemia in patients with type-2 diabetes (T2D), which is due in part to perturbations in pancreatic ꞵ-cell Ca
2+ homeostasis. TALK-1 K+ channels are key regulators of ꞵ-cell electrical excitability, Ca2+ handling and glucose-stimulated insulin secretion (GSIS). KCNK16, which encodes TALK-1 is the most abundant ꞵ-cell K+ channel transcript and also the most islet-restricted ion channel transcript. Furthermore, a nonsynonymous coding sequence polymorphism in KCNK16 causes a predisposition for developing T2D. However, the ability to utilize TALK-1 as a therapeutic target to normalize ꞵ-cell Ca2+ homeostasis in T2D has not been determined. Here, we optimized a high throughput fluorescence-based thallium (TI+ ) flux assay (z' =0.64) to screen for TALK-1 inhibitors, which resulted in identification of 286 TALK-1 inhibitors with potent drug-like properties (from a 50,000 compound screen). Of these hits, 78 exhibit TALK-1 selectivity and do not influence the activity of other K+ channels including TALK-2, TREK-2, TASK-1, TASK-3, KATP or hERG channels. We then performed dose response curve analyses and identified 9 potent inhibitors with IC50 s < 2.5μM that inhibit TALK-1 activity (>65%). To test the efficacy of the inhibitors for their ability to augment islet function, we assessed their impact on mouse islet Ca2+ handling. TALK-1 inhibitors significantly increased islet Ca2+ oscillation frequency (e.g., VU0233408, 2.47 peaks/min vs. DMSO, 1.82 peaks/min, p-value= 0.011), similar to what was previously observed in TALK-1 KO islets. Moreover, the inhibitors did not increase glucose-stimulated Ca2+ oscillation frequency in TALK-1 KO islets. Thus, pharmacological inhibition of TALK-1 is predicted to increase GSIS. In conclusion, we have identified the first potent and selective TALK-1 inhibitors that can be utilized to assess the role of TALK-1 during human ꞵ-cell GSIS under physiological and diabetic conditions. Disclosure: A.Y. Nakhe: None. P. Dadi: None. D. Westover: None. E. Days: None. J. Denton: None. D. Jacobson: None. Funding: National Institute of Diabetes and Digestive and Kidney Diseases (R01DK115620VDC); Vanderbilt University Diabetes and Research Training Center (P60DK20593) [ABSTRACT FROM AUTHOR]- Published
- 2020
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18. An insecticide resistance-breaking mosquitocide targeting inward rectifier potassium channels in vectors of Zika virus and malaria.
- Author
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Swale, Daniel R., Engers, Darren W., Bollinger, Sean R., Gross, Aaron, Inocente, Edna Alfaro, Days, Emily, Kanga, Fariba, Johnson, Reed M., Yang, Liu, Bloomquist, Jeffrey R., Hopkins, Corey R., Piermarini, Peter M., and Denton, Jerod S.
- Abstract
Insecticide resistance is a growing threat to mosquito control programs around the world, thus creating the need to discover novel target sites and target-specific compounds for insecticide development. Emerging evidence suggests that mosquito inward rectifier potassium (Kir) channels represent viable molecular targets for developing insecticides with new mechanisms of action. Here we describe the discovery and characterization of VU041, a submicromolar-affinity inhibitor of Anopheles (An.) gambiae and Aedes (Ae.) aegypti Kir1 channels that incapacitates adult female mosquitoes from representative insecticide-susceptible and -resistant strains of An. gambiae (G3 and Akron, respectively) and Ae. aegypti (Liverpool and Puerto Rico, respectively) following topical application. VU041 is selective for mosquito Kir channels over several mammalian orthologs, with the exception of Kir2.1, and is not lethal to honey bees. Medicinal chemistry was used to develop an analog, termed VU730, which retains activity toward mosquito Kir1 but is not active against Kir2.1 or other mammalian Kir channels. Thus, VU041 and VU730 are promising chemical scaffolds for developing new classes of insecticides to combat insecticide-resistant mosquitoes and the transmission of mosquito-borne diseases, such as Zika virus, without harmful effects on humans and beneficial insects. [ABSTRACT FROM AUTHOR]
- Published
- 2016
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19. Identification and Characterization of a Compound That Protects Cardiac Tissue from Human Ether-à-go-go-related Gene (hERG)-related Drug-induced Arrhythmias.
- Author
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Potet, Franck, Lorinc, Amanda N., Chaigne, Sebastien, Hopkins, Corey R., Venkataraman, Raghav, Stepanovic, Svetlana Z., Lewis, L. Michelle, Days, Emily, Sidorov, Veniamin Y., Engers, Darren W., Beiyan Zou, Afshartous, David, George Jr., Alfred L., Campbell, Courtney M., Balser, Jeffrey R., Min Li, Baudenbacher, Franz J., Lindsley, Craig W., Weaver, C. David, and Kupershmidt, Sabina
- Subjects
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ARRHYTHMIA , *HEART failure , *HEART diseases , *DOFETILIDE , *PATCH-clamp techniques (Electrophysiology) - Abstract
The human Ether-à-go-go-related gene (hERG)-encoded K+ current, IKr is essential for cardiac repolarization but is also a source of cardiotoxicity because unintendedhERGinhibition by diverse pharmaceuticals can cause arrhythmias and sudden cardiac death. We hypothesized that a small molecule that diminishes IKr block by a known hERG antagonist would constitute a first step toward preventing hERG-related arrhythmias and facilitating drug discovery. Using a high-throughput assay, we screened a library of compounds for agents that increase the IC70 of dofetilide, a well characterized hERG blocker. One compound, VU0405601, with the desired activity was further characterized. In isolated, Langendorff-perfused rabbit hearts, optical mapping revealed that dofetilide-induced arrhythmias were reduced after pretreatment with VU0405601. Patch clamp analysis in stable hERG-HEK cells showed effects on current amplitude, inactivation, and deactivation. VU0405601 increased the IC50 of dofetilide from 38.7 to 76.3 nM. VU0405601 mitigates the effects of hERG blockers from the extracellular aspect primarily by reducing inactivation, whereas most clinically relevant hERG inhibitors act at an inner pore site. Structure-activity relationships surrounding VU0405601 identified a 3-pyridiyl and a naphthyridine ring system as key structural components important for preventing hERG inhibition by multiple inhibitors. These findings indicate that small molecules can be designed to reduce the sensitivity of hERG to inhibitors. [ABSTRACT FROM AUTHOR]
- Published
- 2012
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20. Optimization of an ether series of mGlu5 positive allosteric modulators: Molecular determinants of MPEP-site interaction crossover
- Author
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Manka, Jason T., Vinson, Paige N., Gregory, Karen J., Zhou, Ya, Williams, Richard, Gogi, Kiran, Days, Emily, Jadhav, Satya, Herman, Elizabeth J., Lavreysen, Hilde, Mackie, Claire, Bartolomé, José M., Macdonald, Gregor J., Steckler, Thomas, Daniels, J. Scott, Weaver, C. David, Niswender, Colleen M., Jones, Carrie K., Conn, P. Jeffrey, and Lindsley, Craig W.
- Subjects
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GLUTAMATE receptors , *ALLOSTERIC regulation , *ETHERS , *AMIDES , *LABORATORY rodents , *PSYCHOSES , *CHIRALITY - Abstract
Abstract: We report the optimization of a series of non-MPEP site metabotropic glutamate receptor 5 (mGlu5) positive allosteric modulators (PAMs) based on a simple acyclic ether series. Modifications led to a gain of MPEP site interaction through incorporation of a chiral amide in conjunction with a nicotinamide core. A highly potent PAM, 8v (VU0404251), was shown to be efficacious in a rodent model of psychosis. These studies suggest that potent PAMs within topologically similar chemotypes can be developed to preferentially interact or not interact with the MPEP allosteric binding site. [Copyright &y& Elsevier]
- Published
- 2012
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21. Discovery and optimization of a novel, selective and brain penetrant M1 positive allosteric modulator (PAM): The development of ML169, an MLPCN probe
- Author
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Reid, Paul R., Bridges, Thomas M., Sheffler, Douglas J., Cho, Hyekyung P., Lewis, L. Michelle, Days, Emily, Daniels, J. Scott, Jones, Carrie K., Niswender, Colleen M., Weaver, C. David, Conn, P. Jeffrey, Lindsley, Craig W., and Wood, Michael R.
- Subjects
- *
DRUG development , *ALLOSTERIC regulation , *ACETYLCHOLINE , *ALZHEIMER'S disease , *MUSCARINIC receptors , *MOLECULAR probes , *STRUCTURE-activity relationship in pharmacology - Abstract
Abstract: This Letter describes a chemical lead optimization campaign directed at VU0108370, a weak M1 PAM hit with a novel chemical scaffold from a functional HTS screen within the MLPCN. An iterative parallel synthesis approach rapidly established SAR for this series and afforded VU0405652 (ML169), a potent, selective and brain penetrant M1 PAM with an in vitro profile comparable to the prototypical M1 PAM, BQCA, but with an improved brain to plasma ratio. [Copyright &y& Elsevier]
- Published
- 2011
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22. Discovery and Characterization of VU0542270, the First Selective Inhibitor of Vascular Kir6.1/SUR2B K ATP Channels.
- Author
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Li K, McClenahan SJ, Han C, Bungard JD, Rathnayake U, Boutaud O, Bauer JA, Days EL, Lindsley CW, Shelton EL, and Denton JS
- Subjects
- Animals, Mice, Glyburide, Muscle, Smooth, Vascular metabolism, Sulfonylurea Receptors antagonists & inhibitors, KATP Channels antagonists & inhibitors
- Abstract
Vascular smooth muscle K
ATP channels critically regulate blood flow and blood pressure by modulating vascular tone and therefore represent attractive drug targets for treating several cardiovascular disorders. However, the lack of potent inhibitors that can selectively inhibit Kir6.1/SUR2B (vascular KATP ) over Kir6.2/SUR1 (pancreatic KATP ) has eluded discovery despite decades of intensive research. We therefore screened 47,872 chemically diverse compounds for novel inhibitors of heterologously expressed Kir6.1/SUR2B channels. The most potent inhibitor identified in the screen was an N -aryl- N '-benzyl urea compound termed VU0542270. VU0542270 inhibits Kir6.1/SUR2B with an IC50 of approximately 100 nM but has no apparent activity toward Kir6.2/SUR1 or several other members of the Kir channel family at doses up to 30 µM (>300-fold selectivity). By expressing different combinations of Kir6.1 or Kir6.2 with SUR1, SUR2A, or SUR2B, the VU0542270 binding site was localized to SUR2. Initial structure-activity relationship exploration around VU0542270 revealed basic texture related to structural elements that are required for Kir6.1/SUR2B inhibition. Analysis of the pharmacokinetic properties of VU0542270 showed that it has a short in vivo half-life due to extensive metabolism. In pressure myography experiments on isolated mouse ductus arteriosus vessels, VU0542270 induced ductus arteriosus constriction in a dose-dependent manner similar to that of the nonspecific KATP channel inhibitor glibenclamide. The discovery of VU0542270 provides conceptual proof that SUR2-specific KATP channel inhibitors can be developed using a molecular target-based approach and offers hope for developing cardiovascular therapeutics targeting Kir6.1/SUR2B. SIGNIFICANCE STATEMENT: Small-molecule inhibitors of vascular smooth muscle KATP channels might represent novel therapeutics for patent ductus arteriosus, migraine headache, and sepsis; however, the lack of selective channel inhibitors has slowed progress in these therapeutic areas. Here, this study describes the discovery and characterization of the first vascular-specific KATP channel inhibitor, VU0542270., (Copyright © 2024 by The Author(s).)- Published
- 2024
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23. A selective inhibitor of the sperm-specific potassium channel SLO3 impairs human sperm function.
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Lyon M, Li P, Ferreira JJ, Lazarenko RM, Kharade SV, Kramer M, McClenahan SJ, Days E, Bauer JA, Spitznagel BD, Weaver CD, Borrego Alvarez A, Puga Molina LC, Lybaert P, Khambekar S, Liu A, Lindsley CW, Denton J, and Santi CM
- Subjects
- Humans, Male, Membrane Potentials physiology, Semen, Spermatozoa physiology, Infertility, Male, Large-Conductance Calcium-Activated Potassium Channels antagonists & inhibitors
- Abstract
To fertilize an oocyte, the membrane potential of both mouse and human sperm must hyperpolarize (become more negative inside). Determining the molecular mechanisms underlying this hyperpolarization is vital for developing new contraceptive methods and detecting causes of idiopathic male infertility. In mouse sperm, hyperpolarization is caused by activation of the sperm-specific potassium (K
+ ) channel SLO3 [C. M. Santi et al. , FEBS Lett. 584 , 1041-1046 (2010)]. In human sperm, it has long been unclear whether hyperpolarization depends on SLO3 or the ubiquitous K+ channel SLO1 [N. Mannowetz, N. M. Naidoo, S. A. S. Choo, J. F. Smith, P. V. Lishko, Elife 2 , e01009 (2013), C. Brenker et al. , Elife 3 , e01438 (2014), and S. A. Mansell, S. J. Publicover, C. L. R. Barratt, S. M. Wilson, Mol. Hum. Reprod. 20 , 392-408 (2014)]. In this work, we identified the first selective inhibitor for human SLO3-VU0546110-and showed that it completely blocked heterologous SLO3 currents and endogenous K+ currents in human sperm. This compound also prevented sperm from hyperpolarizing and undergoing hyperactivated motility and induced acrosome reaction, which are necessary to fertilize an egg. We conclude that SLO3 is the sole K+ channel responsible for hyperpolarization and significantly contributes to the fertilizing ability of human sperm. Moreover, SLO3 is a good candidate for contraceptive development, and mutation of this gene is a possible cause of idiopathic male infertility.- Published
- 2023
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24. VU6036720: The First Potent and Selective In Vitro Inhibitor of Heteromeric Kir4.1/5.1 Inward Rectifier Potassium Channels.
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McClenahan SJ, Kent CN, Kharade SV, Isaeva E, Williams JC, Han C, Terker A, Gresham R 3rd, Lazarenko RM, Days EL, Romaine IM, Bauer JA, Boutaud O, Sulikowski GA, Harris R, Weaver CD, Staruschenko A, Lindsley CW, and Denton JS
- Subjects
- Animals, Gene Library, High-Throughput Screening Assays, Mice, Potassium metabolism, Potassium Channel Blockers pharmacology, Potassium Channels, Inwardly Rectifying metabolism
- Abstract
Heteromeric Kir4.1/Kir5.1 ( KCNJ10 / KCNJ16 ) inward rectifier potassium (Kir) channels play key roles in the brain and kidney, but pharmacological tools for probing their physiology and therapeutic potential have not been developed. Here, we report the discovery, in a high-throughput screening of 80,475 compounds, of the moderately potent and selective inhibitor VU0493690, which we selected for characterization and chemical optimization. VU0493690 concentration-dependently inhibits Kir4.1/5.1 with an IC
50 of 0.96 μ M and exhibits at least 10-fold selectivity over Kir4.1 and ten other Kir channels. Multidimensional chemical optimization of VU0493690 led to the development of VU6036720, the most potent (IC50 = 0.24 μ M) and selective (>40-fold over Kir4.1) Kir4.1/5.1 inhibitor reported to date. Cell-attached patch single-channel recordings revealed that VU6036720 inhibits Kir4.1/5.1 activity through a reduction of channel open-state probability and single-channel current amplitude. Elevating extracellular potassium ion by 20 mM shifted the IC50 6.8-fold, suggesting that VU6036720 is a pore blocker that binds in the ion-conduction pathway. Mutation of the "rectification controller" asparagine 161 to glutamate (N161E), which is equivalent to small-molecule binding sites in other Kir channels, led to a strong reduction of inhibition by VU6036720. Renal clearance studies in mice failed to show a diuretic response that would be consistent with inhibition of Kir4.1/5.1 in the renal tubule. Drug metabolism and pharmacokinetics profiling revealed that high VU6036720 clearance and plasma protein binding may prevent target engagement in vivo. In conclusion, VU6036720 represents the current state-of-the-art Kir4.1/5.1 inhibitor that should be useful for probing the functions of Kir4.1/5.1 in vitro and ex vivo. SIGNIFICANCE STATEMENT: Heteromeric inward rectifier potassium (Kir) channels comprising Kir4.1 and Kir5.1 subunits play important roles in renal and neural physiology and may represent inhibitory drug targets for hypertension and edema. Herein, we employ high-throughput compound library screening, patch clamp electrophysiology, and medicinal chemistry to develop and characterize the first potent and specific in vitro inhibitor of Kir4.1/5.1, VU6036720, which provides proof-of-concept that drug-like inhibitors of this channel may be developed., (Copyright © 2022 by The American Society for Pharmacology and Experimental Therapeutics.)- Published
- 2022
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25. Challenges in the Discovery and Optimization of mGlu 2/4 Heterodimer Positive Allosteric Modulators.
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Fulton MG, Loch MT, Cuoco CA, Rodriguez AL, Days E, Vinson PN, Kozek KA, Weaver CD, Blobaum AL, Conn PJ, Niswender CM, and Lindsley CW
- Abstract
Background: This article describes the challenges in the discovery and optimization of mGlu2/4 heterodimer Positive Allosteric Modulators (PAMs)., Methods: Initial forays based on VU0155041, a PAM of both the mGlu4 homodimer and the mGlu2/4 heterodimer, led to flat, intractable SAR that precluded advancement. Screening of a collection of 1,152 FDA approved drugs led to the discovery that febuxostat, an approved xanthine oxidase inhibitor, was a moderately potent PAM of the mGlu
2/4 heterodimer (EC50 = 3.4 μM), but was peripherally restricted (rat Kp = 0.03). Optimization of this hit led to PAMs with improved potency (EC50 s <800 nM) and improved CNS penetration (rat Kp >2 , an ~100-fold increase)., Results: However, these new amide analogs of febuxostat proved to be either GIRK1/2 and GIRK1/4 activators (primary carboxamide congeners) or mGlu2 PAMs (secondary and tertiary amides) and not selective mGlu2/4 heterodimer PAMs., Conclusion: These results required the team to develop a new screening cascade paradigm, and exemplified the challenges in developing allosteric ligands for heterodimeric receptors., (© 2019 Bentham Science Publishers.)- Published
- 2019
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26. Discovery, Characterization, and Effects on Renal Fluid and Electrolyte Excretion of the Kir4.1 Potassium Channel Pore Blocker, VU0134992.
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Kharade SV, Kurata H, Bender AM, Blobaum AL, Figueroa EE, Duran A, Kramer M, Days E, Vinson P, Flores D, Satlin LM, Meiler J, Weaver CD, Lindsley CW, Hopkins CR, and Denton JS
- Subjects
- Animals, Binding Sites, Diuretics chemistry, Electrolytes, HEK293 Cells, Humans, Male, Models, Molecular, Molecular Docking Simulation, Molecular Structure, Mutagenesis, Site-Directed, Potassium Channels, Inwardly Rectifying genetics, Rats, Small Molecule Libraries chemistry, Substrate Specificity, Potassium Channels, Inwardly Rectifying antagonists & inhibitors, Potassium Channels, Inwardly Rectifying chemistry, Small Molecule Libraries administration & dosage, Small Molecule Libraries pharmacology
- Abstract
The inward rectifier potassium (Kir) channel Kir4.1 ( KCNJ10 ) carries out important physiologic roles in epithelial cells of the kidney, astrocytes in the central nervous system, and stria vascularis of the inner ear. Loss-of-function mutations in KCNJ10 lead to EAST/SeSAME syndrome, which is characterized by epilepsy, ataxia, renal salt wasting, and sensorineural deafness. Although genetic approaches have been indispensable for establishing the importance of Kir4.1 in the normal function of these tissues, the availability of pharmacological tools for acutely manipulating the activity of Kir4.1 in genetically normal animals has been lacking. We therefore carried out a high-throughput screen of 76,575 compounds from the Vanderbilt Institute of Chemical Biology library for small-molecule modulators of Kir4.1. The most potent inhibitor identified was 2-(2-bromo-4-isopropylphenoxy)- N -(2,2,6,6-tetramethylpiperidin-4-yl)acetamide (VU0134992). In whole-cell patch-clamp electrophysiology experiments, VU0134992 inhibits Kir4.1 with an IC
50 value of 0.97 µ M and is 9-fold selective for homomeric Kir4.1 over Kir4.1/5.1 concatemeric channels (IC50 = 9 µ M) at -120 mV. In thallium (Tl+ ) flux assays, VU0134992 is greater than 30-fold selective for Kir4.1 over Kir1.1, Kir2.1, and Kir2.2; is weakly active toward Kir2.3, Kir6.2/SUR1, and Kir7.1; and is equally active toward Kir3.1/3.2, Kir3.1/3.4, and Kir4.2. This potency and selectivity profile is superior to Kir4.1 inhibitors amitriptyline, nortriptyline, and fluoxetine. Medicinal chemistry identified components of VU0134992 that are critical for inhibiting Kir4.1. Patch-clamp electrophysiology, molecular modeling, and site-directed mutagenesis identified pore-lining glutamate 158 and isoleucine 159 as critical residues for block of the channel. VU0134992 displayed a large free unbound fraction ( fu ) in rat plasma ( fu = 0.213). Consistent with the known role of Kir4.1 in renal function, oral dosing of VU0134992 led to a dose-dependent diuresis, natriuresis, and kaliuresis in rats. Thus, VU0134992 represents the first in vivo active tool compound for probing the therapeutic potential of Kir4.1 as a novel diuretic target for the treatment of hypertension., (Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.)- Published
- 2018
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27. Selective Small Molecule Activators of TREK-2 Channels Stimulate Dorsal Root Ganglion c-Fiber Nociceptor Two-Pore-Domain Potassium Channel Currents and Limit Calcium Influx.
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Dadi PK, Vierra NC, Days E, Dickerson MT, Vinson PN, Weaver CD, and Jacobson DA
- Subjects
- Action Potentials drug effects, Animals, Antibodies pharmacology, Dinoprostone analogs & derivatives, Dinoprostone pharmacology, Electric Stimulation, Fluoxetine pharmacology, HEK293 Cells, Humans, Lectins metabolism, Mice, Mice, Inbred C57BL, Mutation genetics, Potassium Channel Blockers pharmacology, Potassium Channels, Tandem Pore Domain genetics, Potassium Channels, Tandem Pore Domain immunology, Protein Synthesis Inhibitors pharmacology, Tetracycline pharmacology, Action Potentials physiology, Calcium metabolism, Ganglia, Spinal cytology, Nociceptors drug effects, Nociceptors metabolism, Potassium Channels, Tandem Pore Domain metabolism
- Abstract
The two-pore-domain potassium (K2P) channel TREK-2 serves to modulate plasma membrane potential in dorsal root ganglia c-fiber nociceptors, which tunes electrical excitability and nociception. Thus, TREK-2 channels are considered a potential therapeutic target for treating pain; however, there are currently no selective pharmacological tools for TREK-2 channels. Here we report the identification of the first TREK-2 selective activators using a high-throughput fluorescence-based thallium (Tl
+ ) flux screen (HTS). An initial pilot screen with a bioactive lipid library identified 11-deoxy prostaglandin F2α as a potent activator of TREK-2 channels (EC50 ≈ 0.294 μM), which was utilized to optimize the TREK-2 Tl+ flux assay (Z' = 0.752). A HTS was then performed with 76 575 structurally diverse small molecules. Many small molecules that selectively activate TREK-2 were discovered. As these molecules were able to activate single TREK-2 channels in excised membrane patches, they are likely direct TREK-2 activators. Furthermore, TREK-2 activators reduced primary dorsal root ganglion (DRG) c-fiber Ca2+ influx. Interestingly, some of the selective TREK-2 activators such as 11-deoxy prostaglandin F2α were found to inhibit the K2P channel TREK-1. Utilizing chimeric channels containing portions of TREK-1 and TREK-2, the region of the TREK channels that allows for either small molecule activation or inhibition was identified. This region lies within the second pore domain containing extracellular loop and is predicted to play an important role in modulating TREK channel activity. Moreover, the selective TREK-2 activators identified in this HTS provide important tools for assessing human TREK-2 channel function and investigating their therapeutic potential for treating chronic pain.- Published
- 2017
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28. Development and validation of a thallium flux-based functional assay for the sodium channel NaV1.7 and its utility for lead discovery and compound profiling.
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Du Y, Days E, Romaine I, Abney KK, Kaufmann K, Sulikowski G, Stauffer S, Lindsley CW, and Weaver CD
- Subjects
- Dose-Response Relationship, Drug, HEK293 Cells, Humans, Patch-Clamp Techniques, Sodium Channel Blockers chemistry, Veratridine pharmacology, Drug Discovery methods, High-Throughput Screening Assays methods, NAV1.7 Voltage-Gated Sodium Channel metabolism, Sodium Channel Blockers pharmacology, Thallium metabolism
- Abstract
Ion channels are critical for life, and they are targets of numerous drugs. The sequencing of the human genome has revealed the existence of hundreds of different ion channel subunits capable of forming thousands of ion channels. In the face of this diversity, we only have a few selective small-molecule tools to aid in our understanding of the role specific ion channels in physiology which may in turn help illuminate their therapeutic potential. Although the advent of automated electrophysiology has increased the rate at which we can screen for and characterize ion channel modulators, the technique's high per-measurement cost and moderate throughput compared to other high-throughput screening approaches limit its utility for large-scale high-throughput screening. Therefore, lower cost, more rapid techniques are needed. While ion channel types capable of fluxing calcium are well-served by low cost, very high-throughput fluorescence-based assays, other channel types such as sodium channels remain underserved by present functional assay techniques. In order to address this shortcoming, we have developed a thallium flux-based assay for sodium channels using the NaV1.7 channel as a model target. We show that the assay is able to rapidly and cost-effectively identify NaV1.7 inhibitors thus providing a new method useful for the discovery and profiling of sodium channel modulators.
- Published
- 2015
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29. Identification of positive allosteric modulators VU0155094 (ML397) and VU0422288 (ML396) reveals new insights into the biology of metabotropic glutamate receptor 7.
- Author
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Jalan-Sakrikar N, Field JR, Klar R, Mattmann ME, Gregory KJ, Zamorano R, Engers DW, Bollinger SR, Weaver CD, Days EL, Lewis LM, Utley TJ, Hurtado M, Rigault D, Acher F, Walker AG, Melancon BJ, Wood MR, Lindsley CW, Conn PJ, Xiang Z, Hopkins CR, and Niswender CM
- Subjects
- Acetanilides chemistry, Acetanilides pharmacology, Animals, Benzoates pharmacology, CHO Cells, Calcium metabolism, Cricetulus, Dose-Response Relationship, Drug, Excitatory Postsynaptic Potentials drug effects, G Protein-Coupled Inwardly-Rectifying Potassium Channels genetics, G Protein-Coupled Inwardly-Rectifying Potassium Channels metabolism, Glutamic Acid pharmacology, Glycine analogs & derivatives, Glycine pharmacology, HEK293 Cells, Hippocampus drug effects, Hippocampus metabolism, Humans, In Vitro Techniques, Male, Mice, Inbred C57BL, Picolinic Acids chemistry, Picolinic Acids pharmacology, Propionates pharmacology, Pyrroles chemistry, Pyrroles pharmacology, Rats, Receptors, Metabotropic Glutamate genetics, Structure-Activity Relationship, Thallium metabolism, Transfection, Excitatory Amino Acid Agents chemistry, Excitatory Amino Acid Agents pharmacology, Receptors, Metabotropic Glutamate metabolism
- Abstract
Metabotropic glutamate receptor 7 (mGlu7) is a member of the group III mGlu receptors (mGlus), encompassed by mGlu4, mGlu6, mGlu7, and mGlu8. mGlu7 is highly expressed in the presynaptic active zones of both excitatory and inhibitory synapses, and activation of the receptor regulates the release of both glutamate and GABA. mGlu7 is thought to be a relevant therapeutic target for a number of neurological and psychiatric disorders, and polymorphisms in the GRM7 gene have been linked to autism, depression, ADHD, and schizophrenia. Here we report two new pan-group III mGlu positive allosteric modulators, VU0155094 and VU0422288, which show differential activity at the various group III mGlus. Additionally, both compounds show probe dependence when assessed in the presence of distinct orthosteric agonists. By pairing studies of these nonselective compounds with a synapse in the hippocampus that expresses only mGlu7, we have validated activity of these compounds in a native tissue setting. These studies provide proof-of-concept evidence that mGlu7 activity can be modulated by positive allosteric modulation, paving the way for future therapeutics development.
- Published
- 2014
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30. Discovery of (S)-2-cyclopentyl-N-((1-isopropylpyrrolidin2-yl)-9-methyl-1-oxo-2,9-dihydro-1H-pyrrido[3,4-b]indole-4-carboxamide (VU0453379): a novel, CNS penetrant glucagon-like peptide 1 receptor (GLP-1R) positive allosteric modulator (PAM).
- Author
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Morris LC, Nance KD, Gentry PR, Days EL, Weaver CD, Niswender CM, Thompson AD, Jones CK, Locuson CW, Morrison RD, Daniels JS, Niswender KD, and Lindsley CW
- Subjects
- Allosteric Regulation drug effects, Animals, Catalepsy chemically induced, Catalepsy drug therapy, Central Nervous System Agents therapeutic use, Drug Synergism, Exenatide, Glucagon-Like Peptide 1 pharmacology, Glucagon-Like Peptide-1 Receptor, Haloperidol, High-Throughput Screening Assays, Indoles metabolism, Indoles pharmacokinetics, Indoles pharmacology, Insulin metabolism, Insulin Secretion, Islets of Langerhans drug effects, Male, Mice, Inbred C57BL, Microsomes, Liver metabolism, Peptides pharmacology, Pyrrolidines metabolism, Pyrrolidines pharmacokinetics, Pyrrolidines pharmacology, Structure-Activity Relationship, Venoms pharmacology, Indoles chemical synthesis, Pyrrolidines chemical synthesis, Receptors, Glucagon drug effects
- Abstract
A duplexed, functional multiaddition high throughput screen and subsequent iterative parallel synthesis effort identified the first highly selective and CNS penetrant glucagon-like peptide-1R (GLP-1R) positive allosteric modulator (PAM). PAM (S)-9b potentiated low-dose exenatide to augment insulin secretion in primary mouse pancreatic islets, and (S)-9b alone was effective in potentiating endogenous GLP-1R to reverse haloperidol-induced catalepsy.
- Published
- 2014
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31. Dihydrothiazolopyridone derivatives as a novel family of positive allosteric modulators of the metabotropic glutamate 5 (mGlu5) receptor.
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Bartolomé-Nebreda JM, Conde-Ceide S, Delgado F, Iturrino L, Pastor J, Pena MÁ, Trabanco AA, Tresadern G, Wassvik CM, Stauffer SR, Jadhav S, Gogi K, Vinson PN, Noetzel MJ, Days E, Weaver CD, Lindsley CW, Niswender CM, Jones CK, Conn PJ, Rombouts F, Lavreysen H, Macdonald GJ, Mackie C, and Steckler T
- Subjects
- Allosteric Regulation drug effects, Animals, Behavior, Animal drug effects, Dose-Response Relationship, Drug, Humans, Locomotion drug effects, Male, Rats, Receptor, Metabotropic Glutamate 5 metabolism, Structure-Activity Relationship, Antipsychotic Agents chemistry, Antipsychotic Agents pharmacology, Receptor, Metabotropic Glutamate 5 chemistry, Thiazoles chemistry, Thiazoles pharmacology
- Abstract
Starting from a singleton chromanone high throughput screening (HTS) hit, we describe a focused medicinal chemistry optimization effort leading to the identification of a novel series of phenoxymethyl-dihydrothiazolopyridone derivatives as selective positive allosteric modulators (PAMs) of the metabotropic glutamate 5 (mGlu5) receptor. These dihydrothiazolopyridones potentiate receptor responses in recombinant systems. In vitro and in vivo drug metabolism and pharmacokinetic (DMPK) evaluation allowed us to select compound 16a for its assessment in a preclinical animal screen of possible antipsychotic activity. 16a was able to reverse amphetamine-induced hyperlocomotion in rats in a dose-dependent manner without showing any significant motor impairment or overt neurological side effects at comparable doses. Evolution of our medicinal chemistry program, structure activity, and properties relationships (SAR and SPR) analysis as well as a detailed profile for optimized mGlu5 receptor PAM 16a are described.
- Published
- 2013
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32. ML297 (VU0456810), the first potent and selective activator of the GIRK potassium channel, displays antiepileptic properties in mice.
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Kaufmann K, Romaine I, Days E, Pascual C, Malik A, Yang L, Zou B, Du Y, Sliwoski G, Morrison RD, Denton J, Niswender CM, Daniels JS, Sulikowski GA, Xie XS, Lindsley CW, and Weaver CD
- Subjects
- Animals, Anticonvulsants administration & dosage, Anticonvulsants chemistry, Anticonvulsants pharmacology, Calcium Signaling drug effects, Dose-Response Relationship, Drug, Drug Evaluation, Preclinical, Electroshock adverse effects, HEK293 Cells, High-Throughput Screening Assays, Humans, Injections, Intraperitoneal, Mice, Microsomes, Liver metabolism, Molecular Structure, Patch-Clamp Techniques, Pentylenetetrazole toxicity, Phenylurea Compounds administration & dosage, Phenylurea Compounds chemistry, Phenylurea Compounds pharmacology, Pyrazoles administration & dosage, Pyrazoles chemistry, Pyrazoles pharmacology, Rats, Receptors, Metabotropic Glutamate drug effects, Recombinant Proteins drug effects, Seizures etiology, Valproic Acid therapeutic use, Anticonvulsants therapeutic use, G Protein-Coupled Inwardly-Rectifying Potassium Channels agonists, Phenylurea Compounds therapeutic use, Pyrazoles therapeutic use, Seizures drug therapy
- Abstract
The G-protein activated, inward-rectifying potassium (K(+)) channels, "GIRKs", are a family of ion channels (Kir3.1-Kir3.4) that has been the focus of intense research interest for nearly two decades. GIRKs are comprised of various homo- and heterotetrameric combinations of four different subunits. These subunits are expressed in different combinations in a variety of regions throughout the central nervous system and in the periphery. The body of GIRK research implicates GIRK in processes as diverse as controlling heart rhythm, to effects on reward/addiction, to modulation of response to analgesics. Despite years of GIRK research, very few tools exist to selectively modulate GIRK channels' activity and until now no tools existed that potently and selectively activated GIRKs. Here we report the development and characterization of the first truly potent, effective, and selective GIRK activator, ML297 (VU0456810). We further demonstrate that ML297 is active in two in vivo models of epilepsy, a disease where up to 40% of patients remain with symptoms refractory to present treatments. The development of ML297 represents a truly significant advancement in our ability to selectively probe GIRK's role in physiology as well as providing the first tool for beginning to understand GIRK's potential as a target for a diversity of therapeutic indications.
- Published
- 2013
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33. Functional impact of allosteric agonist activity of selective positive allosteric modulators of metabotropic glutamate receptor subtype 5 in regulating central nervous system function.
- Author
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Noetzel MJ, Rook JM, Vinson PN, Cho HP, Days E, Zhou Y, Rodriguez AL, Lavreysen H, Stauffer SR, Niswender CM, Xiang Z, Daniels JS, Jones CK, Lindsley CW, Weaver CD, and Conn PJ
- Subjects
- Allosteric Regulation, Animals, Antipsychotic Agents, Astrocytes, Cell Line, Central Nervous System drug effects, Humans, Mice, Neurons, Receptor, Metabotropic Glutamate 5, Central Nervous System physiology, Receptors, Metabotropic Glutamate agonists
- Abstract
Positive allosteric modulators (PAMs) of metabotropic glutamate receptor subtype 5 (mGlu(5)) have emerged as an exciting new approach for the treatment of schizophrenia and other central nervous system (CNS) disorders. Of interest, some mGlu(5) PAMs act as pure PAMs, only potentiating mGlu(5) responses to glutamate whereas others [allosteric agonists coupled with PAM activity (ago-PAMs)] potentiate responses to glutamate and have intrinsic allosteric agonist activity in mGlu(5)-expressing cell lines. All mGlu(5) PAMs previously shown to have efficacy in animal models act as ago-PAMs in cell lines, raising the possibility that allosteric agonist activity is critical for in vivo efficacy. We have now optimized novel mGlu(5) pure PAMs that are devoid of detectable agonist activity and structurally related mGlu(5) ago-PAMs that activate mGlu(5) alone in cell lines. Studies of mGlu(5) PAMs in cell lines revealed that ago-PAM activity is dependent on levels of mGlu(5) receptor expression in human embryonic kidney 293 cells, whereas PAM potency is relatively unaffected by levels of receptor expression. Furthermore, ago-PAMs have no agonist activity in the native systems tested, including cortical astrocytes and subthalamic nucleus neurons and in measures of long-term depression at the hippocampal Schaffer collateral-CA1 synapse. Finally, studies with pure PAMs and ago-PAMs chemically optimized to provide comparable CNS exposure revealed that both classes of mGlu(5) PAMs have similar efficacy in a rodent model predictive of antipsychotic activity. These data suggest that the level of receptor expression influences the ability of mGlu(5) PAMs to act as allosteric agonists in vitro and that ago-PAM activity observed in cell-based assays may not be important for in vivo efficacy.
- Published
- 2012
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34. Identification and optimization of small molecules that restore E-cadherin expression and reduce invasion in colorectal carcinoma cells.
- Author
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Stoops SL, Pearson AS, Weaver C, Waterson AG, Days E, Farmer C, Brady S, Weaver CD, Beauchamp RD, and Lindsley CW
- Subjects
- Cadherins genetics, Cell Line, Tumor, Drug Evaluation, Preclinical methods, Epithelial-Mesenchymal Transition, Humans, RNA, Messenger metabolism, Cadherins biosynthesis, Colorectal Neoplasms metabolism, Colorectal Neoplasms pathology, Neoplasm Invasiveness prevention & control
- Abstract
E-cadherin is a transmembrane protein that maintains intercellular contacts and cell polarity in epithelial tissue. The down-regulation of E-cadherin contributes to the induction of the epithelial-to-mesenchymal transition (EMT), resulting in an increased potential for cellular invasion of surrounding tissues and entry into the bloodstream. Loss of E-cadherin has been observed in a variety of human tumors as a result of somatic mutations, chromosomal deletions, silencing of the CDH1 gene promoter, and proteolytic cleavage. To date, no compounds directly targeting E-cadherin restoration have been developed. Here, we report the development and use of a novel high-throughput immunofluorescent screen to discover lead compounds that restore E-cadherin expression in the SW620 colon adenocarcinoma cell line. We confirmed restoration of E-cadherin using immunofluorescent microscopy and were able to determine the EC(50) for selected compounds using an optimized In-Cell Western assay. The profiled compounds were also shown to have a minimal effect on cell proliferation but did decrease cellular invasion. We have also conducted preliminary investigations to elucidate a discrete molecular target to account for the phenotypic behavior of these small molecules and have noted a modest increase in E-cadherin mRNA transcripts, and RNA-Seq analysis demonstrated that potent analogues elicited a 10-fold increase in CDH1 (E-cadherin) gene expression.
- Published
- 2011
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35. Discovery of novel allosteric modulators of metabotropic glutamate receptor subtype 5 reveals chemical and functional diversity and in vivo activity in rat behavioral models of anxiolytic and antipsychotic activity.
- Author
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Rodriguez AL, Grier MD, Jones CK, Herman EJ, Kane AS, Smith RL, Williams R, Zhou Y, Marlo JE, Days EL, Blatt TN, Jadhav S, Menon UN, Vinson PN, Rook JM, Stauffer SR, Niswender CM, Lindsley CW, Weaver CD, and Conn PJ
- Subjects
- Allosteric Regulation drug effects, Allosteric Regulation physiology, Animals, Anti-Anxiety Agents chemistry, Anti-Anxiety Agents therapeutic use, Antipsychotic Agents chemistry, Antipsychotic Agents therapeutic use, Cells, Cultured, Cricetinae, Dose-Response Relationship, Drug, HEK293 Cells, Humans, Male, Mice, Motor Activity drug effects, Motor Activity physiology, Psychomotor Agitation physiopathology, Rats, Rats, Sprague-Dawley, Receptor, Metabotropic Glutamate 5, Anti-Anxiety Agents pharmacology, Antipsychotic Agents pharmacology, Psychomotor Agitation drug therapy, Receptors, Metabotropic Glutamate chemistry, Receptors, Metabotropic Glutamate physiology
- Abstract
Modulators of metabotropic glutamate receptor subtype 5 (mGluR5) may provide novel treatments for multiple central nervous system (CNS) disorders, including anxiety and schizophrenia. Although compounds have been developed to better understand the physiological roles of mGluR5 and potential usefulness for the treatment of these disorders, there are limitations in the tools available, including poor selectivity, low potency, and limited solubility. To address these issues, we developed an innovative assay that allows simultaneous screening for mGluR5 agonists, antagonists, and potentiators. We identified multiple scaffolds that possess diverse modes of activity at mGluR5, including both positive and negative allosteric modulators (PAMs and NAMs, respectively). 3-Fluoro-5-(3-(pyridine-2-yl)-1,2,4-oxadiazol-5-yl)benzonitrile (VU0285683) was developed as a novel selective mGluR5 NAM with high affinity for the 2-methyl-6-(phenylethynyl)-pyridine (MPEP) binding site. VU0285683 had anxiolytic-like activity in two rodent models for anxiety but did not potentiate phencyclidine-induced hyperlocomotor activity. (4-Hydroxypiperidin-1-yl)(4-phenylethynyl)phenyl)methanone (VU0092273) was identified as a novel mGluR5 PAM that also binds to the MPEP site. VU0092273 was chemically optimized to an orally active analog, N-cyclobutyl-6-((3-fluorophenyl)ethynyl)nicotinamide hydrochloride (VU0360172), which is selective for mGluR5. This novel mGluR5 PAM produced a dose-dependent reversal of amphetamine-induced hyperlocomotion, a rodent model predictive of antipsychotic activity. Discovery of structurally and functionally diverse allosteric modulators of mGluR5 that demonstrate in vivo efficacy in rodent models of anxiety and antipsychotic activity provide further support for the tremendous diversity of chemical scaffolds and modes of efficacy of mGluR5 ligands. In addition, these studies provide strong support for the hypothesis that multiple structurally distinct mGluR5 modulators have robust activity in animal models that predict efficacy in the treatment of CNS disorders.
- Published
- 2010
- Full Text
- View/download PDF
36. Discovery of N-Aryl Piperazines as Selective mGlu(5) Potentiators with Efficacy in a Rodent Model Predictive of Anti-Psychotic Activity.
- Author
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Zhou Y, Manka JT, Rodriguez AL, Weaver CD, Days EL, Vinson PN, Jadhav S, Hermann EJ, Jones CK, Conn PJ, Lindsley CW, and Stauffer SR
- Abstract
This Letter describes the discovery, SAR and in vitro and in vivo pharmacological profile of a novel non-MPEP derived mGlu(5) positive allosteric modulator (PAM) based upon an N-aryl piperazine chemotype. This mGlu(5) chemotype exhibits the ability to act as either a non-competitive antagonist/negative allosteric modulator (NAM) or potentiator of the glutamate response depending on the identity of the amide substituent, i.e., a 'molecular switch'. A rapidly optimized PAM, 10e (VU0364289), was shown to be potent and specific for the rat mGlu(5) receptor and subsequently demonstrated to be efficacious in a clinically relevant rodent model predictive of anti-psychotic activity, thus providing the first example of a centrally active mGluR(5) PAM optimized from an HTS-derived mGluR5 competitive antagonist.
- Published
- 2010
- Full Text
- View/download PDF
37. Discovery and characterization of novel allosteric potentiators of M1 muscarinic receptors reveals multiple modes of activity.
- Author
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Marlo JE, Niswender CM, Days EL, Bridges TM, Xiang Y, Rodriguez AL, Shirey JK, Brady AE, Nalywajko T, Luo Q, Austin CA, Williams MB, Kim K, Williams R, Orton D, Brown HA, Lindsley CW, Weaver CD, and Conn PJ
- Subjects
- Allosteric Regulation drug effects, Allosteric Regulation physiology, Animals, Binding, Competitive drug effects, Binding, Competitive physiology, CHO Cells, Cells, Cultured, Cricetinae, Cricetulus, Dose-Response Relationship, Drug, Humans, Muscarinic Agonists pharmacology, Rats, Muscarinic Agonists chemistry, Muscarinic Agonists metabolism, Receptor, Muscarinic M1 agonists, Receptor, Muscarinic M1 metabolism
- Abstract
Activators of M(1) muscarinic acetylcholine receptors (mAChRs) may provide novel treatments for schizophrenia and Alzheimer's disease. Unfortunately, the development of M(1)-active compounds has resulted in nonselective activation of the highly related M(2) to M(5) mAChR subtypes, which results in dose-limiting side effects. Using a functional screening approach, we identified several novel ligands that potentiated agonist activation of M(1) with low micromolar potencies and induced 5-fold or greater leftward shifts of the acetylcholine (ACh) concentration-response curve. These ligands did not compete for binding at the ACh binding site, indicating that they modulate receptor activity by binding to allosteric sites. The two most selective compounds, cyclopentyl 1,6-dimethyl-4-(6-nitrobenzo[d][1,3]-dioxol-5-yl)-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate (VU0090157) and (E)-2-(4-ethoxyphenylamino)-N'-((2-hydroxynaphthalen-1-yl)methylene)acetohydrazide (VU0029767), induced progressive shifts in ACh affinity at M(1) that were consistent with their effects in a functional assay, suggesting that the mechanism for enhancement of M(1) activity by these compounds is by increasing agonist affinity. These compounds were strikingly different, however, in their ability to potentiate responses at a mutant M(1) receptor with decreased affinity for ACh and in their ability to affect responses of the allosteric M(1) agonist, 1-[1'-(2-tolyl)-1,4'-bipiperidin-4-yl]-1,3-dihydro-2H-benzimidazol-2-one. Furthermore, these two compounds were distinct in their abilities to potentiate M(1)-mediated activation of phosphoinositide hydrolysis and phospholipase D. The discovery of multiple structurally distinct positive allosteric modulators of M(1) is an exciting advance in establishing the potential of allosteric modulators for selective activation of this receptor. These data also suggest that structurally diverse M(1) potentiators may act by distinct mechanisms and differentially regulate receptor coupling to downstream signaling pathways.
- Published
- 2009
- Full Text
- View/download PDF
38. Discovery, characterization, and antiparkinsonian effect of novel positive allosteric modulators of metabotropic glutamate receptor 4.
- Author
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Niswender CM, Johnson KA, Weaver CD, Jones CK, Xiang Z, Luo Q, Rodriguez AL, Marlo JE, de Paulis T, Thompson AD, Days EL, Nalywajko T, Austin CA, Williams MB, Ayala JE, Williams R, Lindsley CW, and Conn PJ
- Subjects
- Allosteric Regulation, Animals, Antiparkinson Agents administration & dosage, Antiparkinson Agents chemistry, Antiparkinson Agents pharmacology, CHO Cells, Corpus Striatum drug effects, Cricetinae, Cricetulus, Humans, In Vitro Techniques, Injections, Intraventricular, Male, Rats, Rats, Sprague-Dawley, Structure-Activity Relationship, Antiparkinson Agents therapeutic use, Parkinson Disease drug therapy, Receptors, Metabotropic Glutamate drug effects
- Abstract
Parkinson's disease (PD) is caused by the death of dopamine neurons in the basal ganglia and results in motor symptoms such as tremor and bradykinesia. Activation of metabotropic glutamate receptor 4 (mGluR4) has been shown to modulate neurotransmission in the basal ganglia and results in antiparkinsonian effects in rodent PD models. N-Phenyl-7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxamide (PHCCC) is a positive allosteric modulator (PAM) of mGluR4 that has been used to further validate the role of mGluR4 in PD, but the compound suffers from a lack of selectivity, relatively low potency, and poor solubility. Via high-throughput screening, we discovered more than 400 novel PAMs of mGluR4. Compounds derived from a novel chemical scaffold were characterized in vitro at both rat and human mGluR4 using two distinct assays of mGluR4 function. The lead compound was approximately 8-fold more potent than PHCCC, enhanced the potency of glutamate at mGluR4 by 8-fold, and did not show any significant potentiator or antagonist activity at other mGluR subtypes. Resolution of the regioisomers of the lead revealed that the cis regioisomer, (+/-)-cis-2-(3,5-dichlorphenylcarbamoyl)cyclohexanecarboxylic acid (VU0155041), contained the majority of the mGluR4 PAM activity and also exhibited partial agonist activity at mGluR4 at a site that was distinct from the glutamate binding site, suggesting that this compound is a mixed allosteric agonist/PAM of mGluR4. VU0155041 was soluble in an aqueous vehicle, and intracerebroventricular administration of 31 to 316 nmol of VU0155041 dose-dependently decreased haloperidol-induced catalepsy and reserpine-induced akinesia in rats. These exciting results provide continued support for mGluR4 as a therapeutic target in PD.
- Published
- 2008
- Full Text
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39. Age-related loss of neuronal nicotinic receptor expression in the aging mouse hippocampus corresponds with cyclooxygenase-2 and PPAR gamma expression and is altered by long-term NS398 administration.
- Author
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Gahring LC, Persiyanov K, Days EL, and Rogers SW
- Subjects
- Animals, Cyclooxygenase 2, Cyclooxygenase 2 Inhibitors, Cyclooxygenase Inhibitors administration & dosage, Mice, Mice, Inbred C57BL, Neurons drug effects, Neurons metabolism, PPAR gamma genetics, Prostaglandin-Endoperoxide Synthases genetics, Time Factors, Aging drug effects, Aging metabolism, Hippocampus drug effects, Hippocampus metabolism, Nitrobenzenes administration & dosage, PPAR gamma biosynthesis, Prostaglandin-Endoperoxide Synthases biosynthesis, Receptors, Nicotinic biosynthesis, Sulfonamides administration & dosage
- Abstract
Age-related changes in the mammalian dorsal hippocampus are associated with diminished expression of neuronal nicotinic acetylcholine receptors (nAChR), which is particularly severe in pathologies such as those associated with dementias, including Alzheimer's disease. Because the mouse is a useful model for age-related decline in nAChR expression in the basal forebrain and limbic system, we used immunohistochemistry to examine the influence of long-term (12-month) oral administration of nicotine and/or the cyclooxygenase-2 (COX-2) preferring non-steroidal anti-inflammatory drug (NSAID) NS398 on nAChR alpha4, alpha5, alpha7, and beta4 expression in the C57BL/6 mouse. Inhibitory neurons of the dorsal hippocampus that express nAChRs also constitutively express COX-2 and the peroxisome proliferator-antagonist receptor subtype gamma-2 (PPAR gamma2) which is also a target of NS398. Administration of NS398 correlated with retention of nAChR alpha4 and to a lesser extent nAChR beta4, but not nAChR alpha5 or alpha7, but nicotine exhibited no similar effect. Nicotine and NS398 co-administration abolished the NS398-related effect on nAChR alpha4 retention. These results provide evidence that the interaction during aging between oral administration of nicotine and NSAIDs are not straightforward and could even be antagonistic when combined., (Copyright 2004 Wiley Periodicals, Inc.)
- Published
- 2005
- Full Text
- View/download PDF
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