Gregory R. J. Thatcher

Cognitive Deficits in Rats after Forebrain Cholinergic Depletion are Reversed by a Novel NO Mimetic Nitrate Ester

Many conditions adversely affecting learning, memory, and cognition are associated with reductions in forebrain acetylcholine (ACh), most notably aging and Alzheimers disease. In the current study, we demonstrate that bilateral depletion of neocortical and hippocampal ACh in rats produces deficits in a spatial learning task and in a recently described, delayed visual matching-to-sample task. Oral administration of the novel nitrate, GT1061 (4-methyl-5-(2-nitroxyethyl) thiazole HCl), and the acetylcholinesterase inhibitor, donepezil, reversed the cognitive deficits in both memory tasks in a dose-dependent manner. GT1061 was superior in the delayed matching-to-sample task. GT1061 was absorbed rapidly after oral administration, crossed the blood brain barrier, and achieved brain concentrations that were slightly higher than those found in plasma. The activity of GT1061 was NO mimetic: soluble guanylyl cyclase (sGC) was activated, but selectivity was observed for sGC in the hippocampus relative to the vasculature; and hippocampal levels of phosphorylated ERK1/2, which is a postulated intermediary in the formation of long-term memory, were increased. The beneficial effect on visual and spatial memory task performance supports the concept that stimulating the NO/sGC/cGMP signal transduction system can provide new, effective treatments for cognitive disorders. This approach may be superior to that of current drugs that attempt only to salvage the residual function of damaged cholinergic neurons.

Amyloid-β Pathology and APOE Genotype Modulate Retinoid X Receptor Agonist Activity in Vivo

Background: Human APOE effects on RXR agonist activity are unclear. Results: In RXR agonist-treated EFAD mice, beneficial effects in APOE4 hippocampus include ABCA1/ABCG1-induced apoE lipoprotein association/lipidation. Detrimental effects in APOE3 and APOE4 cortex might be from systemic hepatomegaly. Conclusion: Aβ pathology and APOE genotype modulate RXR agonist effects. Significance: Although promising for later stage Alzheimer disease, detrimental side effects limit translational application of RXR agonists. Previous data demonstrate that bexarotene (Bex), retinoid X receptor (RXR) agonist, reduces soluble and insoluble amyloid-β (Aβ) in Alzheimer disease (AD)-transgenic mice either by increasing the levels of mouse apolipoprotein E (apoE) or increasing ABCA1/ABCG1-induced apoE lipoprotein association/lipidation. However, although the mechanism of action of RXR agonists remains unclear, a major concern for their use is human (h)-APOE4, the greatest AD genetic risk factor. If APOE4 imparts a toxic gain-of-function, then increasing apoE4 may increase soluble Aβ, likely the proximal AD neurotoxin. If the APOE4 loss-of-function is lipidation of apoE4, then induction of ABCA1/ABCG1 may be beneficial. In novel EFAD-Tg mice (overexpressing h-Aβ42 with h-APOE), levels of soluble Aβ (Aβ42 and oligomeric Aβ) are highest in E4FAD hippocampus (HP) > E3FAD-HP > E4FAD cortex (CX) > E3FAD-CX, whereas levels of lipoprotein-associated/lipidated apoE have the opposite pattern (6 months). In E4FAD-HP, short-term RXR agonist treatment (Bex or LG100268; 5.75–6 months) increased ABCA1, apoE4 lipoprotein-association/lipidation, and apoE4/Aβ complex, decreased soluble Aβ, and increased PSD95. In addition, hydrogel delivery, which mimics low sustained release, was equally effective as gavage for Bex and LG100268. RXR agonists induced no beneficial effects in the E4FAD-HP in a prevention protocol (5–6 months) and actually increased soluble Aβ levels in E3FAD-CX and E4FAD-CX with the short-term protocol, possibly the result of systemic hepatomegaly. Thus, RXR agonists address the loss-of-function associated with APOE4 and exacerbated by Aβ pathology, i.e. low levels of apoE4 lipoprotein association/lipidation. Further studies are vital to address whether RXR agonists are an APOE4-specific AD therapeutic and the systemic side effects that limit translational application.

Click synthesis of estradiol–cyclodextrin conjugates as cell compartment selective estrogens

Cyclodextrin (CD) is a well known drug carrier and excipient for enhancing aqueous solubility. CDs themselves are anticipated to have low membrane permeability because of relatively high hydrophilicity and molecular weight. CD derivatization with 17-beta estradiol (E(2)) was explored extensively using a number of different click chemistries and the cell membrane permeability of synthetic CD-E(2) conjugate was explored by cell reporter assays and confocal fluorescence microscopy. In simile with reported dendrimer-E(2) conjugates, CD-E(2) was found to be a stable, extranuclear receptor selective estrogen that penetrated into the cytoplasm.

Structure-activity relationships for a family of benzothiophene selective estrogen receptor modulators including raloxifene and arzoxifene.

The search for the “ideal” selective estrogen receptor modulator (SERM) as a substitute for hormone replacement therapy (HRT) or use in cancer chemoprevention has focused on optimization of estrogen receptor (ER) ligand binding. Based on the clinical and preclinical benzothiophene SERMs, raloxifene and arzoxifene, a family of SERMs has been developed to modulate activity and oxidative lability. Antiestrogenic potency measured in human endometrial and breast cancer cells, and ER ligand binding data were correlated and seen to provide a guide to SERM design only when viewed inu2005toto. The inu2005vitro studies were extended to the juvenile rat model, in which the desired antiestrogenic profile and putative cardiovascular benefits of SERMs were observed.

Novel nitrates as NO mimetics directed at Alzheimer's disease

GT 1061 is a novel therapeutic agent that is in Phase 1 clinical studies for Alzheimers disease. GT 1061 is one of a family of novel nitrates that have demonstrated neuroprotective properties and cognition- and memory-enhancing properties in animal models. The prototype of this family, GT 715, has been reported effectively to dissociate the neuromodulatory and the systemic hypotensive effects of nitrates, the latter seriously limiting the therapeutic use of classical nitrates. Further data on the novel nitrates, GT 715 and GT 061, are presented in (a) the malonate-lesion rat model of excitotoxic neurodegeneration, and (b) the reversal of a scopolamine-induced cognition deficit in the Morris water task which tests spatial memory. These data exemplify and reinforce the combined neuroprotective and cognition enhancing properties observed in this family of NO mimetic therapeutic agents. NO mimetics, that mimic the biological activity of NO, will bypass cholinergic receptor activation and are anticipated to provide multiple pathways of treating and circumventing dementia. NO mimetic activation of soluble guanylyl cyclase and cGMP formation in the brain represents one element of an effective neuroprotective strategy. Substantial evidence suggests that NO mimetics may display cGMP-dependent and cGMP-independent activity and may operate via multiple biochemical signaling pathways, both to ensure the survival of neurons subjected to stress and also to provide cognition-enabling pathways to circumvent dementia, providing a combined neuroprotective and cognition-enabling approach to anti-neurodegenerative therapy.

Dimethyl fumarate inhibits the nuclear factor κB pathway in breast cancer cells by covalent modification of p65 protein

In breast tumors, activation of the nuclear factor κB (NFκB) pathway promotes survival, migration, invasion, angiogenesis, stem cell-like properties, and resistance to therapy—all phenotypes of aggressive disease where therapy options remain limited. Adding an anti-inflammatory/anti-NFκB agent to breast cancer treatment would be beneficial, but no such drug is approved as either a monotherapy or adjuvant therapy. To address this need, we examined whether dimethyl fumarate (DMF), an anti-inflammatory drug already in clinical use for multiple sclerosis, can inhibit the NFκB pathway. We found that DMF effectively blocks NFκB activity in multiple breast cancer cell lines and abrogates NFκB-dependent mammosphere formation, indicating that DMF has anti-cancer stem cell properties. In addition, DMF inhibits cell proliferation and significantly impairs xenograft tumor growth. Mechanistically, DMF prevents p65 nuclear translocation and attenuates its DNA binding activity but has no effect on upstream proteins in the NFκB pathway. Dimethyl succinate, the inactive analog of DMF that lacks the electrophilic double bond of fumarate, is unable to inhibit NFκB activity. Also, the cell-permeable thiol N-acetyl l-cysteine, reverses DMF inhibition of the NFκB pathway, supporting the notion that the electrophile, DMF, acts via covalent modification. To determine whether DMF interacts directly with p65, we synthesized and used a novel chemical probe of DMF by incorporating an alkyne functionality and found that DMF covalently modifies p65, with cysteine 38 being essential for the activity of DMF. These results establish DMF as an NFκB inhibitor with anti-tumor activity that may add therapeutic value in the treatment of aggressive breast cancers.

Quinoids Formed from Estrogens and Antiestrogens

Publisher Summary This chapter focuses on the role of quinoids in the DNA damage induced by estrogens and antiestrogen metabolites. Selective estrogen receptor modulators (SERMs) are developed for the treatment of menopause-associated osteoporosis, with a primary application being in the treatment and prevention of breast cancer in postmenopausal women. The most widely used SERM is tamoxifen, which is extensively used for the treatment of hormone-dependent breast cancer and more recently as a chemopreventive agent in women at risk for breast cancer; however, like the estrogens associated with estrogen replacement therapy, tamoxifen can also lead to an increased endometrial cancer risk. The molecular mechanism(s) involved in the carcinogenic action of estrogens and antiestrogens still remains both controversial and elusive. Both can be metabolized to reactive intermediates such as o-quinones and quinone methides, which could cause DNA damage directly through the formation of DNA adducts or indirectly through the generation of reactive oxygen species that oxidize DNA. It is shown that quinones and quinone methides can be formed from both estrogens and antiestrogens. These reactive species can cause damage in cells either through alkylation of cellular macromolecules and/or through generation of reactive oxygen species.

NO-flurbiprofen reduces amyloid β, is neuroprotective in cell culture, and enhances cognition in response to cholinergic blockade

The non‐steroidal anti‐inflammatory drug flurbiprofen is a selective amyloid lowering agent which has been studied clinically in Alzheimer’s disease. HCT‐1026 is an ester prodrug of flurbiprofen incorporating a nitrate carrier moiety that in vivo provides NO bioactivity and an improved safety profile. In vitro, HCT‐1026 retained the cyclooxygenase inhibitory and non‐steroidal anti‐inflammatory drug activity of flurbiprofen, but at concentrations at which levels of amyloid‐β 1–42 amino acid were lowered by flurbiprofen, amyloid‐β 1–42 amino acid levels were elevated 200% by HCT‐1026. Conversely, at lower concentrations, HCT‐1026 behaved as a selective amyloid lowering agent with greater potency than flurbiprofen. The difference in concentration–responses between flurbiprofen and HCT‐1026 in vitro suggests different cellular targets; and in no case did a combination of nitrate drug with flurbiprofen provide similar actions. In vivo, HCT‐1026 was observed to reverse cognitive deficits induced by scopolamine in two behavioral assays; activity that was also shown by a classical nitrate drug, but not by flurbiprofen. The ability to restore aversive memory and spatial working and reference memory after cholinergic blockade has been demonstrated by other agents that stimulate NO/cGMP signaling. These observations add positively to the preclinical profile of HCT‐1026 and NO chimeras in Alzheimer’s disease.

Selective Estrogen Receptor Modulator Delivery of Quinone Warheads to DNA Triggering Apoptosis in Breast Cancer Cells

Estrogen exposure is a risk factor for breast cancer, and estrogen oxidative metabolites have been implicated in chemical carcinogenesis. Oxidation of the catechol metabolite of estrone (4-OHE) and the beta-naphthohydroquinone metabolite of equilenin (4-OHEN) gives o-quinones that produce ROS and damage DNA by adduction and oxidation. To differentiate hormonal and chemical carcinogensis pathways in estrogen receptor positive ER(+) cells, catechol or beta-naphthohydroquinone warheads were conjugated to the selective estrogen receptor modulator (SERM) desmethylarzoxifene (DMA). ER binding was retained in the DMA conjugates; both were antiestrogens with submicromolar potency in mammary and endometrial cells. Cytotoxicity, apoptosis, and caspase-3/7 activation were compared in ER(+) and ER(-)MDA-MB-231 cells, and production of ROS was detected using a fluorescent reporter. Comparison was made to DMA, isolated warheads, and a DMA-mustard. Conjugation of warheads to DMA increased cytotoxicity accompanied by induction of apoptosis and activation of caspase-3/7. Activation of caspase-3/7, induction of apoptosis, and cytotoxicity were all increased significantly in ER(+) cells for the DMA conjugates. ROS production was localized in the nucleus for conjugates in ER(+) cells. Observations are compatible with beta-naphthohydroquinone and catechol groups being concentrated in the nucleus by ER binding, where oxidation and ROS production result, concomitant with caspase-dependent apoptosis. The results suggest that DNA damage induced by catechol estrogen metabolites can be amplified in ER(+) cells independent of hormonal activity. The novel conjugation of quinone warheads to an ER-targeting SERM gives ER-dependent, enhanced apoptosis in mammary cancer cells of potential application in cancer therapy.

Peroxynitrite and NO+ donors form colored nitrite adducts with sinapinic acid: potential applications

Sinapinic acid (3,5-dimethoxy-4-hydroxycinnamic acid, SA) reacted with peroxynitrous acid at neutral pH with a second-order rate constant of 812 M(-1)s(-1), to yield a red product (lambda(max), 532 nm). The identical colored product could be formed with acidified decomposed peroxynitrous acid solutions or nitrite at slower rates (0.1M HCl, 8.32 M(-1)s(-1); 10% acetic acid, 0.0004 M(-1)s(-1)). The red compound is thought to be O-nitrososinapinic acid (3,5-dimethoxy-4-nitrosooxycinnamic acid) which can be formed by reaction with either peroxynitrous acid or nitrous acid. The extinction coefficient of O-nitrososinapinic acid (ONSA) was estimated to be 8419 M(-1)cm(-1) at 510 nm in 10% acetic acid and 90% acetonitrile. ONSA was also formed via NO(+) transfer from S-nitrosoglutathione (GSNO). ONSA in turn can S-nitrosate low molecular weight thiols and protein thiols. SA was also shown to act as a peroxynitrite sink as it effectively prevented the oxidation of dihydrorhodamine under physiological conditions. The fact that O-nitrososinapinic acid is stable and can be used to S-nitrosate thiol containing amino acids, peptides, and proteins makes it a potentially useful reagent in the study of S-nitrosothiol biochemistry and physiology. In addition, the relatively high extinction coefficient of O-nitrososinapinic acid means that it could be utilized as an analyte for the spectroscopic detection of peroxynitrite or NO(+)-donors in the submicromolar range.