Gregory R J Thatcher

University of Illinois at Chicago, Chicago, Illinois, United States

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Publications (105)324.14 Total impact

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    ABSTRACT: The risk of developing hormone-dependent cancers with long-term exposure to estrogens is attributed both to proliferative, hormonal actions at the estrogen receptor (ER), and chemical carcinogenesis elicited by genotoxic, oxidative estrogen metabolites. Non-tumorigenic MCF-10A human breast epithelial cells are classified as ER(-) and undergo estrogen-induced malignant transformation. Selective estrogen receptor modulators (SERMs), in use for breast cancer chemoprevention and for post-menopausal osteoporosis, were observed to inhibit malignant transformation, as measured by anchorage-independent colony growth. This chemopreventive activity was observed to correlate with reduced levels of oxidative estrogen metabolites, cellular ROS, and DNA oxidation. The ability of raloxifene, desmethylarzoxifene (DMA), and bazedoxifene to inhibit this chemical carcinogenesis pathway was not shared by 4-hydroxytamoxifen. Regulation of Phase 2 rather than Phase 1 metabolic enzymes was implicated mechanistically: raloxifene and DMA were observed to upregulate sulfotransferase (SULT 1E1) and glucuronidase (UGT 1A1). The results support upregulation of Phase 2 metabolism in detoxification of catechol estrogen metabolites leading to attenuated ROS formation as a mechanism for inhibition of malignant transformation by a subset of clinically important SERMs.
    Cancer Prevention Research 03/2014; · 4.89 Impact Factor
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    ABSTRACT: Chlormethiazole (CMZ), a clinical sedative/anxiolytic agent, did not reach clinical efficacy in stroke trials despite neuroprotection demonstrated in numerous animal models. Using CMZ as a lead compound, neuroprotective methiazole (MZ) analogues were developed, and neuroprotection and GABAA receptor dependence were studied. Eight MZs were selected from a novel library, of which two were studied in detail. Neuroprotection, glutamate release, intracellular calcium and response to GABA blockade by picrotoxin were measured in rat primary cortical cultures using four cellular models of neurodegeneration. GABA potentiation was assayed in oocytes expressing the α1 β2 γ2 GABAA receptor. Neuroprotection against a range of insults was retained even with substantial chemical modification. Dependence on GABAA receptor activity was variable: at the extremes, neuroprotection by GN-28 was universally sensitive to picrotoxin, while GN-38 was largely insensitive. In parallel, effects on extracellular glutamate and intracellular calcium were associated with GABAA dependence. Consistent with these findings, GN-28 potentiated α1 β2 γ2 GABAA function, whereas GN-38 had weakly inhibitory effect. Neuroprotection against moderate dose oligomeric Aβ1-42 was also tolerant to structural changes. The results support the concept that CMZ does not contain a single pharmacophore, rather that broad-spectrum neuroprotection results from a GABAA -dependent mechanism represented by GN-28, combined with a mechanism represented in GN-38 that shows the least dependence on GABAA receptors. These findings allow further refinement of the neuroprotective pharmacophore and investigation into secondary mechanisms that will assist in identifying methiazole-based compounds of use in treating neurodegeneration.
    British Journal of Pharmacology 10/2013; · 5.07 Impact Factor
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    ABSTRACT: Breast cancer remains a significant cause of death in women, and few therapeutic options exist for estrogen receptor negative (ER (-)) cancers. Epigenetic reactivation of target genes using histone deacetylase (HDAC) inhibitors has been proposed in ER (-) cancers to resensitize to therapy using selective estrogen receptor modulators (SERMs) that are effective in ER (+) cancer treatment. Based upon preliminary studies in ER (+) and ER (-) breast cancer cells treated with combinations of HDAC inhibitors and SERMs, hybrid drugs, termed SERMostats, were designed with computational guidance. Assay for inhibition of four type I HDAC isoforms and antagonism of estrogenic activity in two cell lines yielded a SERMostat with 1-3 μM potency across all targets. The superior hybrid caused significant cell death in ER (-) human breast cancer cells and elicited cell death at the same concentration as the parent SERM in combination treatment and at an earlier time point.
    ChemMedChem 08/2013; · 2.84 Impact Factor
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    ABSTRACT: SELECTIVE ESTROGEN RECEPTOR MODULATORS (SERMS) ARE EFFECTIVE THERAPEUTICS THAT PRESERVE FAVORABLE ACTIONS OF ESTROGENS ON BONE AND ACT AS ANTIESTROGENS IN BREAST TISSUE, DECREASING THE RISK OF VERTEBRAL FRACTURES AND BREAST CANCER, BUT THEIR POTENTIAL IN NEUROPROTECTIVE AND PROCOGNITIVE THERAPY IS LIMITED BY: 1) an increased lifetime risk of thrombotic events; and 2) an attenuated response to estrogens with age, sometimes linked to endothelial nitric oxide synthase (eNOS) dysfunction. Herein, three 3(rd) generation SERMs with similar high affinity for estrogen receptors (ERα, ERβ) were studied: desmethylarzoxifene (DMA), FDMA, and a novel NO-donating SERM (NO-DMA). Neuroprotection was studied in primary rat neurons exposed to oxygen glucose deprivation; reversal of cholinergic cognitive deficit was studied in mice in a behavioral model of memory; long term potentiation (LTP), underlying cognition, was measured in hippocampal slices from older 3×Tg Alzheimer's transgenic mice; vasodilation was measured in rat aortic strips; and anticoagulant activity was compared. Pharmacologic blockade of GPR30 and NOS; denudation of endothelium; measurement of NO; and genetic knockout of eNOS were used to probe mechanism. Comparison of the three chemical probes indicates key roles for GPR30 and eNOS in mediating therapeutic activity. Procognitive, vasodilator and anticoagulant activities of DMA were found to be eNOS dependent, while neuroprotection and restoration of LTP were both shown to be dependent upon GPR30, a G-protein coupled receptor mediating estrogenic function. Finally, the observation that an NO-SERM shows enhanced vasodilation and anticoagulant activity, while retaining the positive attributes of SERMs even in the presence of NOS dysfunction, indicates a potential therapeutic approach without the increased risk of thrombotic events.
    PLoS ONE 01/2013; 8(8):e70740. · 3.73 Impact Factor
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    ABSTRACT: The promising therapeutic potential of the NO-donating hybrid aspirin prodrugs (NO-ASA) includes induction of chemopreventive mechanisms and has been reported in almost 100 publications. One example, NCX-4040 (pNO-ASA), is bioactivated by esterase to a quinone methide (QM) electrophile. In cell cultures, pNO-ASA and QM-donating X-ASA prodrugs that cannot release NO rapidly depleted intracellular GSH and caused DNA damage; however, induction of Nrf2 signaling elicited cellular defense mechanisms including upregulation of NAD(P)H:quinone oxidoreductase-1 (NQO1) and glutamate-cysteine ligase (GCL). In HepG2 cells, the "NO-specific" 4,5-diaminofluorescein reporter, DAF-DA, responded to NO-ASA and X-ASA, with QM-induced oxidative stress masquerading as NO. LC-MS/MS analysis demonstrated efficient alkylation of Cys residues of proteins including glutathione-S-transferase-P1 (GST-P1) and Kelch-like ECH-associated protein 1 (Keap1). Evidence was obtained for alkylation of Keap1 Cys residues associated with Nrf2 translocation to the nucleus, nuclear translocation of Nrf2, activation of antioxidant response element (ARE), and upregulation of cytoprotective target genes. At least in cell culture, pNO-ASA acts as a QM donor, bioactivated by cellular esterase activity to release salicylates, NO(3)(-), and an electrophilic QM. Finally, two novel aspirin prodrugs were synthesized, both potent activators of ARE, designed to release only the QM and salicylates on bioactivation. Current interest in electrophilic drugs acting via Nrf2 signaling suggests that QM-donating hybrid drugs can be designed as informative chemical probes in drug discovery.
    Chemical Research in Toxicology 10/2012; · 3.67 Impact Factor
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    ABSTRACT: Learning and memory deficits in Alzheimer's disease (AD) result from synaptic failure and neuronal loss, the latter caused in part by excitotoxicity and oxidative stress. A therapeutic approach is described that uses NO-chimeras directed at restoration of both synaptic function and neuroprotection. 4-Methylthiazole (MZ) derivatives were synthesized, based upon a lead neuroprotective pharmacophore acting in part by GABA(A) receptor potentiation. MZ derivatives were assayed for protection of primary neurons against oxygen-glucose deprivation and excitotoxicity. Selected neuroprotective derivatives were incorporated into NO-chimera prodrugs, coined nomethiazoles. To provide proof of concept for the nomethiazole drug class, selected examples were assayed for restoration of synaptic function in hippocampal slices from AD-transgenic mice, reversal of cognitive deficits, and brain bioavailability of the prodrug and its neuroprotective MZ metabolite. Taken together, the assay data suggest that these chimeric nomethiazoles may be of use in treatment of multiple components of neurodegenerative disorders, such as AD.
    Journal of Medicinal Chemistry 07/2012; 55(15):6784-801. · 5.61 Impact Factor
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    ABSTRACT: The bioactivation of both endogenous and equine estrogens to electrophilic quinoid metabolites has been postulated as a contributing factor in carcinogenic initiation and/or promotion in hormone sensitive tissues. Bearing structural resemblance to estrogens, extensive studies have shown that many selective estrogen receptor modulators (SERMs) are subject to similar bioactivation pathways. Lasofoxifene (LAS), a third generation SERM which has completed phase III clinical trials for the prevention and treatment of osteoporosis, is currently approved in the European Union for this indication. Previously, Prakash et al. (Drug Metab. Dispos. (2008) 36, 1218-1226) reported that similar to estradiol, two catechol regioisomers of LAS are formed as primary oxidative metabolites, accounting for roughly half of the total LAS metabolism. However, the potential for further oxidation of these catechols to electrophilic o-quinones has not been reported. In the present study, LAS was synthesized and its oxidative metabolism investigated in vitro under various conditions. Incubation of LAS with tyrosinase, human liver microsomes, or rat liver microsomes in the presence of GSH as a trapping reagent resulted in the formation of two mono-GSH and two di-GSH catechol conjugates which were characterized by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Similar conjugates were also detected in incubations with P450 3A4, P450 2D6, and P450 1B1 supersomes. Interestingly, these conjugates were also detected as major metabolites when compared to competing detoxification pathways such as glucuronidation and methylation. The 7-hydroxylasofoxifene (7-OHLAS) catechol regioisomer was also synthesized and oxidized either chemically or enzymatically to an o-quinone that was shown to form depurinating adducts with DNA. Collectively, these data show that analogous to estrogens, LAS is oxidized to catechols and o-quinones which could potentially contribute to in vivo toxicity for this SERM.
    Chemical Research in Toxicology 05/2012; 25(7):1472-83. · 3.67 Impact Factor
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    ABSTRACT: 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.
    Journal of Medicinal Chemistry 03/2012; 55(7):3076-87. · 5.61 Impact Factor
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    ABSTRACT: S-Nitrosothiol (RSNO) formation is one manner by which nitric oxide (•NO) exerts its biological effects. There are several proposed mechanisms of formation of RSNO in vivo: auto-oxidation of •NO, transnitrosation, oxidative nitrosylation, and from dinitrosyliron complexes (DNIC). Both free •NO, generated by •NO donors, and S-nitrosocysteine (CysNO) are widely used to study •NO biology and signaling, including protein S-nitrosation. It is assumed that the cellular effects of both compounds are analogous and indicative of in vivo •NO biology. A quantitative comparison was made of formation of DNIC and RSNO, the major •NO-derived cellular products. In RAW 264.7 cells, both •NO and CysNO were metabolized, leading to rapid intracellular RSNO and DNIC formation. DNIC were the dominant products formed from physiologic •NO concentrations, however, and RSNO were the major product from CysNO treatment. Chelatable iron was necessary for DNIC assembly from either •NO or CysNO, but not for RSNO formation. These profound differences in RSNO and DNIC formation from •NO and CysNO question the use of CysNO as a surrogate for physiologic •NO. Researchers designing experiments intended to elucidate the biological signaling mechanisms of •NO should be aware of these differences and should consider the biological relevance of the use of exogenous CysNO.
    Antioxidants & Redox Signaling 02/2012; 17(7):962-8. · 8.20 Impact Factor
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    ABSTRACT: o-Quinone forming estrogens and selective estrogen receptor modulators (SERMs) have been associated with carcinogenesis. LY2066948, a novel SERM in development by Eli Lilly for the treatment of uterine fibroids and myomas, has structural similarity to the equine estrogen equilenin present in hormone replacement formulations; both contain a naphthol group susceptible to oxidative metabolism to o-quinones. LY2066948 was synthesized and assayed for antiestrogenic activity, and in cell culture was confirmed to be a more potent antiestrogen than the prototypical SERM, 4-hydroxytamoxifen. Oxidation of LY2066948 with 2-iodoxybenzoic acid gave an o-quinone (t(1/2)=3.9 ± 0.1h) which like 4-hydroxyequilenin-o-quinone (t(1/2)=2.5 ± 0.2 h) was observed to be exceptionally long-lived with the potential to cause cytotoxicity and/or genotoxicity. In model reactions with tyrosinase, the catechol metabolites of LY2066948 and equilenin were products; interestingly, in the presence of ascorbate to inhibit autoxidation, these catechols were formed quantitatively. Tyrosinase incubations in the presence of GSH gave the expected GSH conjugates resulting from trapping of the o-quinones, which were characterized by LC-MS/MS. Incubations of LY2066948 or equilenin with rat liver microsomes also gave detectable o-quinone trapped GSH conjugates; however, as observed with other SERMs, oxidative metabolism of LY2066948 mainly occurred on the amino side chain to yield the N-dealkylated metabolite. CYP1B1 is believed to be responsible for extra-hepatic generation of genotoxic estrogen quinones and o-quinone GSH conjugates were detected in equilenin incubations. However, in corresponding incubations with CYP1B1 supersomes, no o-quinone GSH conjugates of LY2066948 were detected. These studies suggest that although the naphthol group is susceptible to oxidative metabolism to long-lived o-quinones, the formation of these quinones by cytochrome P450 can be attenuated by the chemistry of the remainder of the molecule as in the case of LY2066948.
    Chemico-biological interactions 01/2012; 196(1-2):1-10. · 2.46 Impact Factor
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    ABSTRACT: Long-term exposure to estrogens including those in traditional hormone replacement therapy (HRT) increases the risk of developing hormone-dependent cancers. As a result, women are turning to over-the-counter (OTC) botanical dietary supplements, such as black cohosh (Cimicifuga racemosa) and hops (Humulus lupulus), as natural alternatives to HRT. The two major mechanisms which likely contribute to estrogen and/or HRT cancer risk are: the estrogen receptor-mediated hormonal pathway; and the chemical carcinogenesis pathway involving formation of estrogen quinones that damage DNA and proteins, hence initiating and promoting carcinogenesis. Because, OTC botanical HRT alternatives are in widespread use, they may have the potential for chemopreventive effects on estrogen carcinogenic pathways in vivo. Therefore, the effect of OTC botanicals on estrogen-induced malignant transformation of MCF-10A cells was studied. Cytochrome P450 catalyzed hydroxylation of estradiol at the 4-position leads to an o-quinone believed to act as the proximal carcinogen. Liquid chromatography/tandem mass spectrometry analysis of estradiol metabolites showed that 4-hydroxylation was inhibited by hops, whereas black cohosh was without effect. Estrogen-induced expression of CYP450 1B1 and CYP450 1A1 was attenuated by the hops extract. Two phenolic constituents of hops (xanthohumol, XH; 8-prenylnaringenin, 8-PN) were tested: 8-PN was a potent inhibitor, whereas XH had no effect. Finally, estrogen-induced malignant transformation of MCF-10A cells was observed to be significantly inhibited by hops (5 μg/mL) and 8-PN (50 nmol/L). These data suggest that hops extracts possess cancer chemopreventive activity through attenuation of estrogen metabolism mediated by 8-PN.
    Cancer Prevention Research 01/2012; 5(1):73-81. · 4.89 Impact Factor
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    ABSTRACT: Alzheimer's disease (AD) is the most common form of dementia among the elderly with women exhibiting a higher risk than men for the disease. Due to these gender differences, there is great interest in the role that estrogens play in cognitive impairment and the onset of the classic amyloid and tau lesions in AD. Human and rodent studies indicate a strong association between low brain aromatase, sex hormone levels, and beta amyloid deposition. Therefore, the effects of depleting both circulating and brain estrogen levels, through gonadectomy and/or treatment with the aromatase inhibitor, anastrozole, upon hippocampal AD-like pathology in male and female 3xTgAD mice were evaluated. Liquid chromatography-mass spectrometry revealed anastrozole serum levels of 10.19 ng/mL and for the first time brain levels were detected at 4.7 pg/mL. Densitometric analysis of the hippocampus revealed that anastrozole significantly increased Aβ- but not APP/Aβ-immunoreactivity in intact 3xTgAD females compared to controls (p<0.001). Moreover, anastrozole significantly increased the number of Aβ- compared to APP/Aβ-positive hippocampal CA1 neurons in intact and gonadectomized female mice. Concurrently, anastrozole significantly reduced the APP/Aβ plaque load in 9 month old female 3xTgAD mice. These data suggest that anastrozole treatment differentially affects select amyloid species which in turn may play a role in the extraneuronal to intraneuronal deposition of this peptide.
    Neurobiology of Disease 09/2011; 45(1):479-87. · 5.62 Impact Factor
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    ABSTRACT: 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.
    ACS Medicinal Chemistry Letters 09/2011; 2(9):656-661. · 3.31 Impact Factor
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    ABSTRACT: 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.
    ACS Chemical Neuroscience 05/2011; 2(5):256-268. · 3.87 Impact Factor
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    ABSTRACT: 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.
    Journal of Medicinal Chemistry 03/2011; 54(7):2293-306. · 5.61 Impact Factor
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    ABSTRACT: The rearrangement of organic thionitrate to sulfenyl nitrite potentially mediates the release of nitric oxide from organic nitrates, such as nitroglycerin, in the presence of thiol. The biological activity of these nitrovasodilators is proposed to result from release of nitric oxide in vivo. The thionitrate rearrangement bears analogy to the rearrangement of peroxynitrous acid to nitric acid, which has been proposed to mediate the biological toxicity of nitric oxide and superoxide. In this paper, the two concerted rearrangement processes and competing homolytic reactions are explored using molecular orbital calculations at levels up to MP4SDQ/6-31G*//MP2/6-31G*. Examination of structure and energy for all conformers and isomers of RSONO2 (R = H, Me), models for organic thionitrates and their isomers, demonstrates that structures corresponding to thionitrates and sulfenyl nitrates are of similar energy. Free energies of reaction for homolysis of these compounds are low (ΔG0 < 19 kcal/mol), whereas the barrier for concerted rearrangement is large (ΔG≠(aq.) = 56 kcal/mol). The larger barrier for concerted rearrangement of peroxynitrous acid to nitric acid (ΔG≠(aq.) = 60 kcal/mol) again compares unfavourably with homolysis (ΔG0 < 11 kcal/mol for homolysis to NO2 or •NO). The transition state structures, confirmed by normal mode and intrinsic reaction coordinate analysis, indicate that considerable structural reorganization is required for concerted rearrangement of the ground state species. These results suggest that concerted rearrangement is not likely to be a viable step in either biological process. However, rearrangement via homolysis and radical recombination may provide an energetically accessible pathway for peroxynitrous acid rearrangement to nitric acid and rearrangement of thionitrate to sulfenyl nitrite. In this case, NO2 will be a primary product of both reactions. Keywords: thionitrate, nitric oxide, peroxynitrite, nitrovasodilator, nitrate.
    Canadian Journal of Chemistry 02/2011; 73(10):1627-1638. · 0.96 Impact Factor
  • Ronald Kluger, Gregory R. J. Thatcher, William C. Stallings
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    ABSTRACT: Compounds 1–5 were prepared to compare reactivity patterns of cyclic and acyclic phosphonylurea esters. The rates and products of reactions of phosphonylurea esters (1–3) with hydroxide in aqueous acetonitrile were analyzed. In these compounds the phosphonate moiety is in a strained five-membered ring, which also contains the ureido group. Structural determination of 1 by X-ray crystallography indicates that the five-membered ring is planar and the internal ring angle at phosphorus is 93.1°. The endocyclic N—C—N angle of the ureido group is 111°. The compounds undergo hydrolysis in alkaline aqueous acetonitrile at 35 °C with a rate about 106 times that of analogues (4, 5) in which the phosphonate group is exocyclic to the ureido ring. Compound 1 undergoes alkaline hydrolysis (k = 9.0 × 103 M−1 s−1) to release the phenoxy group to give 6. The hydrolysis of alkyl esters 2 (k = 2.4 × 104 M−1 s−1)and 3(k = 1.3 × 103 M−1 s−1) leads to cleavage of the endocyclic P—N bond, producing 7 and 8 respectively. The exocyclic alkyl esters (4 and 5) also cleave at the P—N bond with respective rate constants of 6.5 × 10−3 M−1 s−1 and 4.4 × 10−2 M−1 s−1. The data are consistent with a mechanism in which hydroxide adds to 1 to form a pentacoordinate phosphorus intermediate with the phenoxy group in an equatorial position and the ureido ring in apical and equatorial positions (with nitrogen apical). The departure of the urea group is slower than pseudorotation of the intermediate and expulsion of phenoxide. In the isomerized intermediate, phenoxy is apical but the methylene group of the ring, which has low apicophilicity, must also be apical. Reactions of 2 and 3, which have more basic oxygen leaving groups, occur with P—N cleavage because expulsion from the isomerized intermediate in those cases is not sufficiently fast. These results fit reaction patterns at phosphorus that are determined by ring strain and electronegativity of ligands. Contributions from effects due to antiperiplanar interactions between bonding and nonbonding electrons are not detected.
    Canadian Journal of Chemistry 02/2011; 65(8):1838-1844. · 0.96 Impact Factor
  • Yue-Ting Wang, Sujeewa C. Piyankarage, Gregory R. J. Thatcher
    Free Radical Biology and Medicine - FREE RADICAL BIOL MED. 01/2011; 51.
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    ABSTRACT: There is association between exposure to estrogens and the development and progression of hormone-dependent gynecological cancers. Chemical carcinogenesis by catechol estrogens derived from oxidative metabolism is thought to contribute to breast cancer, yet exact mechanisms remain elusive. Malignant transformation was studied in MCF-10A human mammary epithelial cells, since estrogens are not proliferative in this cell line. The human and equine estrogen components of estrogen replacement therapy (ERT) and their catechol metabolites were studied, along with the influence of co-administration of selective estrogen receptor modulators (SERMs), raloxifene and desmethyl-arzoxifene (DMA), and histone deacetylase inhibitors. Transformation was induced by human estrogens, and selectively by the 4-OH catechol metabolite, and to a lesser extent by an equine estrogen metabolite. The observed estrogen-induced upregulation of CYP450 1B1 in estrogen receptor negative MCF-10A cells, was compatible with a causal role for 4-OH catechol estrogens, as was attenuated transformation by CYP450 inhibitors. Estrogen-induced malignant transformation was blocked by SERMs correlating with a reduction in formation of nucleobase catechol estrogen (NCE) adducts and formation of 8-oxo-dG. NCE adducts can be formed consequent to DNA abasic site formation, but NCE adducts were also observed on incubation of estrogen quinones with free nucleotides. These results suggest that NCE adducts may be a biomarker for cellular electrophilic stress, which together with 8-oxo-dG as a biomarker of oxidative stress correlate with malignant transformation induced by estrogen oxidative metabolites. The observed attenuation of transformation by SERMs correlated with these biomarkers and may also be of clinical significance in breast cancer chemoprevention.
    PLoS ONE 01/2011; 6(11):e27876. · 3.73 Impact Factor
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    ABSTRACT: Estrogen action, via both nuclear and extranuclear estrogen receptors (ERs), induces a variety of cellular signals that are prosurvival or proliferative, whereas nitric oxide (NO) can inhibit apoptosis via caspase S-nitrosylation and via activation of soluble guanylyl cyclase to produce cGMP. The action of 17β-estradiol (E(2)) at ER is known to elicit NO signaling via activation of NO synthase (NOS) in many tissues. The MCF-10A nontumorigenic, mammary epithelial cell line is genetically stable and insensitive to estrogenic proliferation. In this cell line, estrogens or NOS inhibitors alone had no significant effect, whereas in combination, apoptosis was induced rapidly in the absence of serum; the presence of inducible NOS was confirmed by proteomic analysis. The application of pharmacological agents determined that apoptosis was dependent upon NO/cGMP signaling via cyclic GMP (cGMP)-dependent protein kinase and could be replicated by inhibition of the phosphatidylinositol 3 kinase/serine-threonine kinase pathway prior to addition of E(2). Apoptosis was confirmed by nuclear staining and increased caspase-3 activity in E(2) + NOS inhibitor-treated cells. Apoptosis was partially inhibited by a pure ER antagonist and replicated by agonists selective for extranuclear ER. Cells were rescued from E(2)-induced apoptosis after NOS blockade, by NO-donors and cGMP pathway agonists; preincubation with NO donors was required. The NOS and ER status of breast cancer tissues is significant in etiology, prognosis, and therapy. In this study, apoptosis of preneoplastic mammary epithelial cells was triggered by estrogens via a rapid, extranuclear ER-mediated response, after removal of an antiapoptotic NO/cGMP/cGMP-dependent protein kinase signal.
    Endocrinology 10/2010; 151(12):5602-16. · 4.72 Impact Factor

Publication Stats

912 Citations
812 Downloads
324.14 Total Impact Points

Institutions

  • 2003–2013
    • University of Illinois at Chicago
      • • Department of Medicinal Chemistry and Pharmacognosy
      • • College of Pharmacy
      Chicago, Illinois, United States
  • 1991–2007
    • Queen's University
      • • Department of Pharmacology and Toxicology
      • • Department of Chemistry
      Kingston, Ontario, Canada