Publications by authors named "Isaac T Schiefer"

18 Publications

  • Page 1 of 1

E-cigarette aerosols containing nicotine modulate nicotinic acetylcholine receptors and astroglial glutamate transporters in mesocorticolimbic brain regions of chronically exposed mice.

Chem Biol Interact 2021 Jan 23;333:109308. Epub 2020 Nov 23.

Department of Pharmacology and Experimental Therapeutics, College of Pharmacy and Pharmaceutical Sciences, The University of Toledo, Toledo, OH, 43614, USA. Electronic address:

Nicotine exposure increases the release of glutamate in part through stimulatory effects on pre-synaptic nicotinic acetylcholine receptors (nAChRs). To assess the impact of chronic electronic (e)-cigarette use on these drug dependence pathways, we exposed C57BL/6 mice to three types of inhalant exposures for 3 months; 1) e-cigarette aerosol generated from liquids containing nicotine (ECN), 2) e-cigarette aerosol generated from liquids containing vehicle chemicals without nicotine (Veh), and 3) air only (AC). We investigated the effects of daily e-cigarette exposure on protein levels of α7 nAChR and α4/β2 nAChR, gene expression and protein levels of astroglial glutamate transporters, including glutamate transporter-1 (GLT-1) and cystine/glutamate antiporter (xCT), in the frontal cortex (FC), striatum (STR) and hippocampus (HIP). We found that chronic inhalation of ECN increased α4/β2 nAChR in all brain regions, and increased α7 nAChR expression in the FC and STR. The total GLT-1 relative mRNA and protein expression were decreased in the STR. Moreover, GLT-1 isoforms (GLT-1a and GLT-1b) were downregulated in the STR in ECN group. However, inhalation of e-cigarette aerosol downregulated xCT expression in STR and HIP compared to AC and Veh groups. ECN group had increased brain-derived neurotrophic factor in the STR compared to control groups. Finally, mass spectrometry detected high concentrations of the nicotine metabolite, cotinine, in the FC and STR in ECN group. This work demonstrates that chronic inhalation of nicotine within e-cigarette aerosols significantly alters the expression of nAChRs and astroglial glutamate transporters in specific mesocorticolimbic brain regions.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cbi.2020.109308DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7752837PMC
January 2021

Combining Neurobehavioral Analysis and Photoaffinity Labeling to Understand Protein Targets of Methamphetamine in Casper Zebrafish.

ACS Chem Neurosci 2020 09 18;11(17):2761-2773. Epub 2020 Aug 18.

Department of Medicinal and Biological Chemistry, College of Pharmacy and Pharmaceutical Sciences, University of Toledo, Toledo, Ohio 43606, United States.

Photoaffinity labeling (PAL) remains one of the most widely utilized methods of determining protein targets of drugs. Although useful, the scope of this technique has been limited to applications because of the inability of UV light to penetrate whole organisms. Herein, pigment-free Casper zebrafish were employed to allow PAL. A methamphetamine-related phenethylamine PAL probe, designated here as , demonstrated dose-dependent effects on behavior similar to methamphetamine and permitted concentration-dependent labeling of protein binding partners. Click chemistry was used to analyze binding partners via fluoroimaging. Conjugation to a biotin permitted streptavidin pull-down and proteomic analysis to define direct binding partners of the methamphetamine probe. Bioinformatic analysis revealed the probe was chiefly bound to proteins involved in phagocytosis and mitochondrial function. Future applications of this experimental paradigm combining examination of drug-protein binding interactions alongside neurobehavioral readouts via PAL will significantly enhance our understanding of drug targets, mechanism(s) of action, and toxicity/lethality.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acschemneuro.0c00416DOI Listing
September 2020

The Effect of Electronic-Cigarette Vaping on Cardiac Function and Angiogenesis in Mice.

Sci Rep 2019 03 11;9(1):4085. Epub 2019 Mar 11.

Department of Medicine, University of Toledo College of Medicine, Toledo, OH, 43614, USA.

The rapid increase in use of electronic-cigarettes (e-cigarettes), especially among youth, raises the urgency for regulating bodies to make informed decisions, guidance, and policy on these products. This study evaluated cardiac function in an experimental model following exposure to e-cigarettes. We subjected C57BL/6 mice to e-cigarette vaping for 2-weeks, and cardiac function was assessed using echocardiography. Cardiac tissues were collected at the end of e-cigarette exposure for pathological analysis. The experimental data showed that e-cigarette vaping (3 h/day for 14 days) had no significant effect on cardiac contractility as measured by ejection fraction. However, it significantly increased angiogenesis in mouse heart tissue. We found that e-cigarette exposure increased the endothelial cell marker CD31 and CD34 to approximately 2 fold (p < 0.05) in heart tissue from female mice and about 150% (p < 0.05) in male mice. E-cigarette vaping also caused slower weight gain compared to mice exposed to room air. In addition, short-term e-cigarette exposure slightly increased collagen content in heart tissue but did not result in significant tissue fibrosis. These results suggest that short-term exposure to e-cigarettes has no acute effect on cardiac contractile function or tissue fibrosis, but it increases cardiac angiogenesis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-019-40847-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6411855PMC
March 2019

Pharmacokinetics and pharmacodynamics of nitric oxide mimetic agents.

Nitric Oxide 2019 03 11;84:69-78. Epub 2019 Jan 11.

Department of Medicinal and Biological Chemistry, College of Pharmacy and Pharmaceutical Sciences, University of Toledo, Toledo, USA. Electronic address:

Drug discovery focusing on NO mimetics has been hamstrung due to its unconventional nature. Central to these challenges is the fact that direct measurement of molecular NO in biological systems is exceedingly difficulty. Hence, drug development of NO mimetics must rely upon measurement of the NO donating specie (i.e., a prodrug) and a downstream marker of efficacy without directly measuring the molecule, NO, that is responsible for biological effect. The focus of this review is to catalog in vivo attempts to monitor the pharmacokinetics (PK) of the NO donating specie and the pharmacodynamic (PD) readout of NO bioactivity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.niox.2019.01.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6979316PMC
March 2019

Quantification of phenolic acid metabolites in humans by LC-MS: a structural and targeted metabolomics approach.

Bioanalysis 2018 Oct;10(19):1591-1608

Psychiatry Service, Louis Stokes Cleveland Department of Veterans Affairs Medical Center, Cleveland, OH, 44106, USA.

Aim: Co-metabolism between a human host and the gastrointestinal microbiota generates many small phenolic molecules such as 3-hydroxy-3-(3-hydroxyphenyl)propanoic acid (3,3-HPHPA), which are reported to be elevated in schizophrenia and autism. Characterization of these chemicals, however, has been limited by analytic challenges.

Methodology/results: We applied HPLC to separate and quantify over 50 analytes, including multiple structural isomers of 3,3-HPHPA in human cerebrospinal fluid, serum and urine. Confirmation of identity was provided by NMR, by MS and other detection methods. The highly selective methods support rapid quantification of multiple metabolites and exhibit superior chromatographic behavior.

Conclusion: An improved ultra-HPLC-MS/MS and structural approaches can accurately quantify 3,3-HPHPA and related analytes in human biological matrices.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.4155/bio-2018-0140DOI Listing
October 2018

Furoxans (Oxadiazole-4 N-oxides) with Attenuated Reactivity are Neuroprotective, Cross the Blood Brain Barrier, and Improve Passive Avoidance Memory.

J Med Chem 2018 05 7;61(10):4593-4607. Epub 2018 May 7.

Nitric oxide (NO) mimetics and other agents capable of enhancing NO/cGMP signaling have demonstrated efficacy as potential therapies for Alzheimer's disease. A group of thiol-dependent NO mimetics known as furoxans may be designed to exhibit attenuated reactivity to provide slow onset NO effects. The present study describes the design, synthesis, and evaluation of a furoxan library resulting in the identification of a prototype furoxan, 5a, which was profiled for use in the central nervous system. Furoxan 5a demonstrated negligible reactivity toward generic cellular thiols under physiological conditions. Nonetheless, cGMP-dependent neuroprotection was observed, and 5a (20 mg/kg) reversed cholinergic memory deficits in a mouse model of passive avoidance fear memory. Importantly, 5a can be prepared as a pharmaceutically acceptable salt and is observed in the brain 12 h after oral administration, suggesting potential for daily dosing and excellent metabolic stability. Continued investigation into furoxans as attenuated NO mimetics for the CNS is warranted.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.jmedchem.8b00389DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6344890PMC
May 2018

Development of a reactive oxygen species-sensitive nitric oxide synthase inhibitor for the treatment of ischemic stroke.

Free Radic Biol Med 2018 02 22;115:395-404. Epub 2017 Dec 22.

Department of Medicinal and Biological Chemistry, College of Pharmacy and Pharmaceutical Sciences, University of Toledo, OH 43614, USA. Electronic address:

Ischemic stroke is caused by a blockage of cerebral blood flow resulting in neuronal and glial hypoxia leading to inflammatory and reactive oxygen species (ROS)-mediated cell death. Nitric oxide (NO) formed by NO synthase (NOS) is known to be protective in ischemic stroke, however NOS has been shown to 'uncouple' under oxidative conditions to instead produce ROS. Nitrones are antioxidant molecules that are shown to trap ROS to then decompose and release NO. In this study, the nitrone 5 was designed such that its decomposition product is a NOS inhibitor, 6, effectively leading to NOS inhibition specifically at the site of ROS production. The ability of 5 to spin-trap radicals and decompose to 6 was observed using EPR and LC-MS/MS. The pro-drug concept was tested in vitro by measuring cell viability and 6 formation in SH-SY5Y cells subjected to oxygen glucose deprivation (OGD). 5 was found to be more efficacious and more potent than PBN, and was able to increase phospho-Akt while reducing nitrotyrosine and cleaved caspase-3 levels. 6 treatment, but not 5, was found to decrease NO production in LPS-stimulated microglia. Doppler flowmetry on anesthetized mice showed increased cerebral blood flow upon intravenous administration of 1mg/kg of 5, but a return to baseline upon administration of 10mg/kg, likely due to its dual nature of antioxidant/NO-donor and NOS-inhibition. Mice treated with 5 after permanent ischemia exhibited a >30% reduction in infarct volume, and higher formation of 6 in ischemic tissue resulting in region specific effects limited to the infarct area.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.freeradbiomed.2017.12.027DOI Listing
February 2018

Identification and characterization of 5α-cyprinol-sulfating cytosolic sulfotransferases (Sults) in the zebrafish (Danio rerio).

J Steroid Biochem Mol Biol 2017 11 12;174:120-127. Epub 2017 Aug 12.

Department of Pharmacology, College of Pharmacy, University of Toledo Health Science Campus, Toledo, OH 43614, USA. Electronic address:

5α-Cyprinol 27-sulfate is the major biliary bile salt present in cypriniform fish including the zebrafish (Danio rerio). The current study was designed to identify the zebrafish cytosolic sulfotransferase (Sult) enzyme(s) capable of sulfating 5α-cyprinol and to characterize the zebrafish 5α-cyprinol-sulfating Sults in comparison with human SULT2A1. Enzymatic assays using zebrafish homogenates showed 5α-cyprinol-sulfating activity. A systematic analysis, using a panel of recombinant zebrafish Sults, revealed two Sult2 subfamily members, Sult2st2 and Sult2st3, as major 5α-cyprinol-sulfating Sults. Both enzymes showed higher activities using 5α-cyprinol as the substrate, compared to their activity with DHEA, a representative substrate for mammalian SULT2 family members, particularly SULT2A1. pH-Dependence and kinetics experiments indicated that the catalytic properties of zebrafish Sult2 family members in mediating the sulfation of 5α-cyprinol were different from those of either zebrafish Sult3st4 or human SULT2A1. Collectively, these results imply that both Sult2st2 and Sult2st3 have evolved to sulfate specifically C-bile alcohol, 5α-cyprinol, in Cypriniform fish, whereas the enzymatic characteristics of zebrafish Sult3 members, particularly Sult3st4, correlated with those of human SULT2A1.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jsbmb.2017.08.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5675747PMC
November 2017

Effects of chronic inhalation of electronic cigarettes containing nicotine on glial glutamate transporters and α-7 nicotinic acetylcholine receptor in female CD-1 mice.

Prog Neuropsychopharmacol Biol Psychiatry 2017 07 27;77:1-8. Epub 2017 Mar 27.

Department of Pharmacology and Experimental Therapeutics, College of Pharmacy and Pharmaceutical Sciences, The University of Toledo, Toledo, OH 43614, USA. Electronic address:

Alteration in glutamate neurotransmission has been found to mediate the development of drug dependence, including nicotine. We and others, through using western blotting, have reported that exposure to drugs of abuse reduced the expression of glutamate transporter-1 (GLT-1) as well as cystine/glutamate antiporter (xCT), which consequently increased extracellular glutamate concentrations in the mesocorticolimbic area. However, our previous studies did not reveal any changes in glutamate/aspartate transporter (GLAST) following exposure to drugs of abuse. In the present study, for the first time, we investigated the effect of chronic exposure to electronic (e)-cigarette vapor containing nicotine, for one hour daily for six months, on GLT-1, xCT, and GLAST expression in frontal cortex (FC), striatum (STR), and hippocampus (HIP) in outbred female CD1 mice. In this study, we also investigated the expression of alpha-7 nicotinic acetylcholine receptor (α-7 nAChR), a major pre-synaptic nicotinic receptor in the glutamatergic neurons, which regulates glutamate release. We found that inhalation of e-cigarette vapor for six months increased α-7 nAChR expression in both FC and STR, but not in the HIP. In addition, chronic e-cigarette exposure reduced GLT-1 expression only in STR. Moreover, e-cigarette vapor inhalation induced downregulation of xCT in both the STR and HIP. We did not find any significant changes in GLAST expression in any brain region. Finally, using liquid chromatography-tandem mass spectrometry (LC-MS/MS) techniques, we detected high concentrations of nicotine and cotinine, a major metabolite of nicotine, in the FC tissues of e-cigarette exposed mice. These data provide novel evidence about the effects of chronic nicotine inhalation on the expression of key glial glutamate transporters as well as α-7 nAChR. Our work may suggest that nicotine exposure via chronic inhalation of e-cigarette vapor may be mediated in part by alterations in the glutamatergic system.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.pnpbp.2017.03.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5466499PMC
July 2017

Novel Selective Calpain 1 Inhibitors as Potential Therapeutics in Alzheimer's Disease.

J Alzheimers Dis 2016 ;49(3):707-21

Department of Pathology and Cell Biology, The Taub Institute for Research on Alzheimer's Disease and the Aging Brain, Columbia University, New York, NY, USA.

Alzheimer's disease, one of the most important brain pathologies associated with neurodegenerative processes, is related to overactivation of calpain-mediated proteolysis. Previous data showed a compelling efficacy of calpain inhibition against abnormal synaptic plasticity and memory produced by the excess of amyloid-β, a distinctive marker of the disease. Moreover, a beneficial effect of calpain inhibitors in Alzheimer's disease is predictable by the occurrence of calpain hyperactivation leading to impairment of memory-related pathways following abnormal calcium influxes that might ensue independently of amyloid-β elevation. However, molecules currently available as effective calpain inhibitors lack adequate selectivity. This work is aimed at characterizing the efficacy of a novel class of epoxide-based inhibitors, synthesized to display improved selectivity and potency towards calpain 1 compared to the prototype epoxide-based generic calpain inhibitor E64. Both functional and preliminary toxicological investigations proved the efficacy, potency, and safety of the novel and selective calpain inhibitors NYC438 and NYC488 as possible therapeutics against the disease.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3233/JAD-150618DOI Listing
November 2016

Proteasome activation is a mechanism for pyrazolone small molecules displaying therapeutic potential in amyotrophic lateral sclerosis.

ACS Chem Neurosci 2014 Sep 18;5(9):823-9. Epub 2014 Jul 18.

Department of Chemistry, ‡Department of Molecular Biosciences, Rice Institute for Biomedical Research, and §Department of Molecular Biosciences, Chemistry of Life Processes Institute, Center for Molecular Innovation and Drug Discovery, Northwestern University , Evanston, Illinois 60208, United States.

Amyotrophic lateral sclerosis (ALS) is a progressive and ultimately fatal neurodegenerative disease. Pyrazolone containing small molecules have shown significant disease attenuating efficacy in cellular and murine models of ALS. Pyrazolone based affinity probes were synthesized to identify high affinity binding partners and ascertain a potential biological mode of action. Probes were confirmed to be neuroprotective in PC12-SOD1(G93A) cells. PC12-SOD1(G93A) cell lysates were used for protein pull-down, affinity purification, and subsequent proteomic analysis using LC-MS/MS. Proteomics identified the 26S proteasome regulatory subunit 4 (PSMC1), 26S proteasome regulatory subunit 6B (PSMC4), and T-complex protein 1 (TCP-1) as putative protein targets. Coincubation with appropriate competitors confirmed the authenticity of the proteomics results. Activation of the proteasome by pyrazolones was demonstrated in the absence of exogenous proteasome inhibitor and by restoration of cellular protein degradation of a fluorogenic proteasome substrate in PC12-SOD1(G93A) cells. Importantly, supplementary studies indicated that these molecules do not induce a heat shock response. We propose that pyrazolones represent a rare class of molecules that enhance proteasomal activation in the absence of a heat shock response and may have therapeutic potential in ALS.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/cn500147vDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4176317PMC
September 2014

Design, synthesis, and optimization of novel epoxide incorporating peptidomimetics as selective calpain inhibitors.

J Med Chem 2013 Aug 22;56(15):6054-68. Epub 2013 Jul 22.

Department of Medicinal Chemistry and Pharmacognosy, University of Illinois College of Pharmacy, University of Illinois at Chicago, 833 S. Wood Street, Chicago, IL 60612-7231, USA.

Hyperactivation of the calcium-dependent cysteine protease calpain 1 (Cal1) is implicated as a primary or secondary pathological event in a wide range of illnesses and in neurodegenerative states, including Alzheimer's disease (AD). E-64 is an epoxide-containing natural product identified as a potent nonselective, calpain inhibitor, with demonstrated efficacy in animal models of AD. By use of E-64 as a lead, three successive generations of calpain inhibitors were developed using computationally assisted design to increase selectivity for Cal1. First generation analogues were potent inhibitors, effecting covalent modification of recombinant Cal1 catalytic domain (Cal1cat), demonstrated using LC-MS/MS. Refinement yielded second generation inhibitors with improved selectivity. Further library expansion and ligand refinement gave three Cal1 inhibitors, one of which was designed as an activity-based protein profiling probe. These were determined to be irreversible and selective inhibitors by kinetics studies comparing full length Cal1 with the general cysteine protease papain.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/jm4006719DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3962784PMC
August 2013

Furoxans (1,2,5-oxadiazole-N-oxides) as novel NO mimetic neuroprotective and procognitive agents.

J Med Chem 2012 Apr 30;55(7):3076-87. Epub 2012 Mar 30.

Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Illinois at Chicago, (MC 781), 833 South Wood Street, Chicago, Illinois 60612-7231, USA.

Furoxans (1,2,5-oxadiazole-N-oxides) are thiol-bioactivated NO-mimetics that have not hitherto been studied in the CNS. Incorporation of varied substituents adjacent to the furoxan ring system led to modulation of reactivity toward bioactivation, studied by HPLC-MS/MS analysis of reaction products. Attenuated reactivity unmasked the cytoprotective actions of NO in contrast to the cytotoxic actions of higher NO fluxes reported previously for furoxans. Neuroprotection was observed in primary neuronal cell cultures following oxygen glucose deprivation (OGD). Neuroprotective activity was observed to correlate with thiol-dependent bioactivation to produce NO(2)(-), but not with depletion of free thiol itself. Neuroprotection was abrogated upon cotreatment with a sGC inhibitor, ODQ, thus supporting activation of the NO/sGC/CREB signaling cascade by furoxans. Long-term potentiation (LTP), essential for learning and memory, has been shown to be potentiated by NO signaling, therefore, a peptidomimetic furoxan was tested in hippocampal slices treated with oligomeric amyloid-β peptide (Aβ) and was shown to restore synaptic function. The novel observation of furoxan activity of potential therapeutic use in the CNS warrants further studies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/jm201504sDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3407596PMC
April 2012

NO-SSRIs: Nitric Oxide Chimera Drugs Incorporating a Selective Serotonin Reuptake Inhibitor.

ACS Med Chem Lett 2011 Sep;2(9):656-661

Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Illinois at Chicago, 833 S. Wood St., Chicago, IL 60612-7231.

Hybrid nitrate drugs have been reported to provide NO bioactivity to ameliorate side effects or to provide ancillary therapeutic activity. Hybrid nitrate selective serotonin reuptake inhibitors (NO-SSRIs) were prepared to improve the therapeutic profile of this drug class. A synthetic strategy for use of a thiocarbamate linker was developed, which in the case of NO-fluoxetine facilitated hydrolysis to fluoxetine at pH 7.4 within 7 hours. In cell culture, NO-SSRIs were weak inhibitors of the serotonin transporter, however, in the forced swimming task (FST) in rats, NO-fluoxetine demonstrated classical antidepressant activity. Comparison of NO-fluoxetine, with fluoxetine, and an NO-chimera nitrate developed for Alzheimer's disease (GT-1061), was made in the step through passive avoidance (STPA) test of learning and memory in rats treated with scopolamine as an amnesic agent. Fluoxetine was inactive, whereas NO-fluoxetine and GT-1061 both restored long-term memory. GT-1061 also produced antidepressant behavior in FST. These data support the potential for NO-SSRIs to overcome the lag in onset of therapeutic action and provide co-therapy of neuropathologies concomitant with depression.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/ml2000033DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3171965PMC
September 2011

Benzothiophene Selective Estrogen Receptor Modulators Provide Neuroprotection by a novel GPR30-dependent Mechanism.

ACS Chem Neurosci 2011 May;2(5):256-268

Department of Medicinal Chemistry and Pharmacognosy (M/C 781) College of Pharmacy University of Illinois at Chicago 833 South Wood Street Chicago, Illinois 60612-7231.

The clinical benzothiophene SERM (BT-SERM), raloxifene, was compared with estrogens in protection of primary rat neurons against oxygen-glucose deprivation (OGD). Structure-activity relationships for neuroprotection were determined for a family of BT-SERMs displaying a spectrum of ERα and ERβ binding affinity and agonist/antagonist activity, leading to discovery of a neuroprotective pharmacophore, present in the clinically relevant SERMS, raloxifene and desmethylarzoxifene (DMA), for which submicromolar potency was observed for neuroprotection. BT-SERM neuroprotection did not correlate with binding to ER nor classical ER activity, however, both the neuroprotective SERMs and estrogens were shown, using pharmacological probes, to activate the same kinase signaling cascades. The antiestrogen ICI 182,780 inhibited the actions of estrogens, but not those of BT-SERMs, whereas antagonism of the G-protein coupled receptor, GPR30, was effective for both SERMs and estrogens. Since SERMs have antioxidant activity, ER-independent mechanisms were studied using the classical phenolic antioxidants, BHT and Trolox, and the Nrf2-dependent cytoprotective electrophile, sulforaphane. However, neuroprotection by these agents was not sensitive to GPR30 antagonism. Collectively, these data indicate that the activity of neuroprotective BT-SERMs is GPR30-dependent and ER-independent and not mediated by antioxidant effects. Comparison of novel BT-SERM derivatives and analogs identified a neuroprotective pharmacophore of potential use in design of novel neuroprotective agents with a spectrum of ER activity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/cn100106aDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3124785PMC
May 2011

Inhibition of amyloidogenesis by nonsteroidal anti-inflammatory drugs and their hybrid nitrates.

J Med Chem 2011 Apr 15;54(7):2293-306. Epub 2011 Mar 15.

Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Illinois at Chicago, MC 781, 833 South Wood Street, Chicago, Illinois 60612-7231, United States.

Poor blood-brain barrier penetration of nonsteroidal anti-inflammatory drugs (NSAIDs) has been blamed for the failure of the selective amyloid lowering agent (SALA) R-flurbiprofen in phase 3 clinical trials for Alzheimer's disease (AD). NO-donor NSAIDs (NO-NSAIDs) provide an alternative, gastric-sparing approach to NSAID SALAs, which may improve bioavailability. NSAID analogues were studied for anti-inflammatory activity and for SALA activity in N2a neuronal cells transfected with human amyloid precursor protein (APP). Flurbiprofen (1) analogues were obtained with enhanced anti-inflammatory and antiamyloidogenic properties compared to 1, however, esterification led to elevated Aβ(1-42) levels. Hybrid nitrate prodrugs possessed superior anti-inflammatory activity and reduced toxicity relative to the parent NSAIDs, including clinical candidate CHF5074. Although hybrid nitrates elevated Aβ(1-42) at higher concentration, SALA activity was observed at low concentrations (≤1 μM): both Aβ(1-42) and the ratio of Aβ(1-42)/Aβ(1-40) were lowered. This biphasic SALA activity was attributed to the intact nitrate drug. For several compounds, the selective modulation of amyloidogenesis was tested using an immunoprecipitation MALDI-TOF approach. These data support the development of NO-NSAIDs as an alternative approach toward a clinically useful SALA.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/jm101450pDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3072465PMC
April 2011

Proteomic and mass spectroscopic quantitation of protein S-nitrosation differentiates NO-donors.

ACS Chem Biol 2010 Jul;5(7):667-80

Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Illinois at Chicago, Chicago, Illinois 60612-7231, USA.

Protein S-nitrosation has been argued to be the most important signaling pathway mediating the bioactivity of NO. This post-translational modification of protein thiols is the result of chemical nitrosation of cysteine residues. The term NO-donors covers very different chemical classes, from clinical therapeutics to probes of routine use in chemical biology; their different chemistry is predicted to result in distinctive biology regulated by protein S-nitrosation. To measure the extent of protein S-nitrosation by NO-donors, a proteomic mass spectrometry method was developed, which quantitates free thiol versus nitrosothiol for each modified cysteine residue, coined d-Switch. This method is adapted from the biotin switch (BST) method, used extensively to identify S-nitrosated proteins in complex biological mixtures; however, BST does not quantitate free thiol. Since glutathione-S-transferase P1-1 (GST-P1) has been proposed to be a biological "NO-carrier", GST-P1 was used as a reporter protein. The 5 different chemical classes of NO-donors compared by d-Switch demonstrated very different profiles of protein S-nitrosation and response to O(2) and cysteine, although all NO-donors were oxidants toward GST-P1. The low limits of detection and the ability to use established MS database searching allowed facile generalization of the d-Switch method. Therefore after incubation of neuronal cell cultures with nitrosothiol, it was possible to quantitate not only S-nitrosation of GST-P1 but also many other proteins, including novel targets such as ubiquitin carboxyl-terminal esterase L1 (UCHL1). Moreover, d-Switch also allowed identification of non-nitrosated proteins and quantitation of degree of nitrosation for individual protein thiols.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/cb100054mDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2941763PMC
July 2010