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Arabinose 5-phosphate isomerase (API) catalyzes the reversible isomerization of Ribulose 5-phosphate (Ru5P) to Arabinose 5-Phosphate (Ar5P) for the production of 3-deoxy-2-octulosonic acid 8-phosphate (KDO), a component of bacterial lipopolysaccharide (LPS) of gram-negative bacteria. API is an attractive target for therapeutic development against gram-negative bacterial pathogens. The current assay method of API activity utilizes a general reaction for keto sugar determination in a secondary, 3-h color development reaction with 25 N sulfuric acid which poses hazard to both personnel and instrumentation. We therefore aimed to develop a more user friendly assay of the enzyme. Since Ru5P absorbs in the UV region and contains at least 2 chiral centers, it can be expected to display circular dichroism (CD). A wavelength scan revealed indeed Ru5P displays a pronounced negative ellipticity of 30,560 mDeg M−1cm−1 at 279 nm in Tris buffer pH 9.1 but Ar5P does not have any CD. API enzymatic reactions were monitored directly and continuously in real time by following the disappearance of CD from the Ru5P substrate, or by the appearance of CD from Ar5P substrate. The CD signal at this wavelength was not affected by absorption of the enzyme protein or of small molecules, or turbidity of the solution. Common additives in protein and enzyme reaction mixtures such as detergents, metals, and 5% dimethylsulfoxide did not interfere with the CD signal. Assay reactions of 1–3 min consistently yielded reproducible results. Introduction of accessories in a spectropolarimeter will easily adapt this assay to high throughput format for screening thousands of small molecules as inhibitor candidates of API.

The bioactivity of antimetabolites is connected to molecular recognition by the target proteins, which makes the stereochemical configuration of the chiral centers in these molecules, including those in the modifying moieties, of extreme importance. In an attempt to enhance the anti-cancer activity of a newly identified 5-[(R/S-α-tert-butyl-2-nitro-4-{2-phenyl}alkynyl)benzyloxy]methyl-2′-deoxyuridine, initially evaluated as a 1:1 mixture of diastereomers with respect to the modifying moiety, we first took the excision library approach, which revealed the importance of all the structural features of the moiety for bioactivity. We then proceeded with the synthesis of the secondary derivatives, varying the substitution pattern within the moiety. These attempts failed to improve bioactivity. However, examination of individually made stereoisomers revealed a 3-fold potency increase in the S-isomer compared with the diastereomeric mixture, without significantly affecting the toxicity against normal rapidly dividing cells.

Over expression of cysteine proteases in human body causes neurodegenerative diseases, destruction of cartilage tissue, and bone atrophy, and some of them are implicated in destructive role of malignant tumors and cancer metastasis. Several non-human cysteine proteases play a key role in life cycles of certain foreign invasive organisms. Therefore, inhibition of cysteine proteases represents an important venue for finding potential therapeutic agents against Alzheimer's disease, multiple sclerosis, ischemic stroke, myocardial infarcts, carcinoma progression, as well as some parasital and viral infections. Affinity labeling agents used as inhibitors of cysteine proteases normally bear an electrophilic "warhead", a reactive group that covalently binds the active site cysteine residue thereby inactivating the enzyme, but achieving both high activity and selectivity remains challenging. We are developing cyclopropene derivatives that show selective binding of thiol residues of cysteine. Several derivatives of cyclopropenes have been synthesized and evaluated as potential cysteine-binding "warheads". Their stability in aqueous media and reactivity toward cysteine and other amino acid chemical probes was examined. We have found that 1,2-cyclopropene moiety irreversibly binds the thiol group of cysteine, leaving other amino acid residues unaffected, which has the advantage for targeting enzymes expressed in foreign organisms or promoting carcinoma progression.

Background Nitric oxide (NO) has been heavily implicated in migraine. Nitroglycerin is a prototypic NO-donor, and triggers migraine in humans. However, nitroglycerin also induces oxidative/nitrosative stress and is a source of peroxynitrite – factors previously linked with migraine etiology. Soluble guanylyl cyclase (sGC) is the high affinity NO receptor in the body, and the aim of this study was to identify the precise role of sGC in acute and chronic migraine. Methods We developed a novel brain-bioavailable sGC stimulator (VL-102), and tested its hyperalgesic properties in mice. We also determined the effect of VL-102 on c-fos and calcitonin gene related peptide (CGRP) immunoreactivity within the trigeminovascular complex. In addition, we also tested the known sGC inhibitor, ODQ, within the chronic nitroglycerin migraine model. Results VL-102-evoked acute and chronic mechanical cephalic and hind-paw allodynia in a dose-dependent manner, which was blocked by the migraine medications sumatriptan, propranolol, and topiramate. In addition, VL-102 also increased c-fos and CGRP expressing cells within the trigeminovascular complex. Importantly, ODQ completely inhibited acute and chronic hyperalgesia induced by nitroglycerin. ODQ also blocked hyperalgesia already established by chronic nitroglycerin, implicating this pathway in migraine chronicity. Conclusions These results indicate that nitroglycerin causes migraine-related pain through stimulation of the sGC pathway, and that super-activation of this receptor may be an important component for the maintenance of chronic migraine. This work opens the possibility for negative sGC modulators as novel migraine therapies.

Electrophilic reactive intermediates resulting from drug metabolism have been associated with toxicity and off-target effects and in some drug discovery programs trigger NO-GO decisions. Many botanicals and dietary supplements are replete with such reactive electrophiles, notably Michael acceptors, which have been demonstrated to elicit chemo-preventive mechanisms; and Michael acceptors are gaining regulatory approval as contemporary cancer therapeutics. Identifying protein targets of these electrophiles is central to understanding potential therapeutic benefit and toxicity risk. NO-donating NSAID prodrugs (NO-NSAIDs) have been the focus of extensive clinical and preclinical studies in inflammation and cancer chemoprevention and therapy: a subset exemplified by pNO-ASA, induces chemopreventive mechanisms following bioactivation to an electrophilic quinone methide (QM) Michael acceptor. Having previously shown that these NO-independent, QM-donors activated Nrf2 via covalent modification of Keap-1, we demonstrate that components of canonical NF-κB signaling are also targets, leading to the inhibition of NF-κB signaling. Combining bio-orthogonal probes of QM-donor ASA prodrugs with mass spectrometric proteomics and pathway analysis, we proceeded to characterize the quinonome: the protein cellular targets of QM-modification by pNO-ASA and its ASA pro-drug congeners. Further comparison was made using a biorthogonal probe of the “bare-bones”, Michael acceptor, and clinical anti-inflammatory agent, dimethyl fumarate, which we have shown to inhibit NF-κB signaling. Identified quinonome pathways include post-translational protein folding, cell-death regulation, protein transport, and glycolysis; and identified proteins included multiple heat shock elements, the latter functionally confirmed by demonstrating activation of heat shock response.

We recently identified a genome-wide genetic association of eosinophilic esophagitis (EoE) at 2p23 spanning the calpain 14 (CAPN14) gene, yet the causal mechanism has not been elucidated. We now show that recombinant CAPN14 cleaves a calpain-specific substrate and is inhibited by 4 classical calpain inhibitors: MDL-28170, acetyl-calpastatin, E-64, and PD151746. CAPN14 is specifically induced (>100-fold) in esophageal epithelium after IL-13 treatment. Epithelial cells overexpressing CAPN14 display impaired epithelial architecture, characterized by acantholysis, epidermal clefting, and epidermolysis. CAPN14 overexpression impairs epithelial barrier function, as demonstrated by decreased transepithelial resistance (2.1-fold) and increased FITC-dextran flux (2.6-fold). Epithelium with gene-silenced CAPN14 demonstrates increased dilated intercellular spaces (5.5-fold) and less organized basal cell layering (1.5-fold) following IL-13 treatment. Finally, CAPN14 overexpression results in loss of desmoglein 1 (DSG1) expression, whereas the IL-13-induced loss of DSG1 is normalized by CAPN14 gene silencing. Importantly, these findings were specific to CAPN14, as they were not observed with modulation of CAPN1 expression. These results, along with the potent induction of CAPN14 by IL-13 and genetic linkage of EoE to the CAPN14 gene locus, demonstrate a molecular and cellular pathway that contributes to T helper type 2 responses in mucosal epithelium.

The Human Genome Project has facilitated the sequencing of many species, yet the current Sanger method is too expensive, labor intensive and time consuming to accomplish medical resequencing of human genomes en masse. Of the 'next-generation' technologies, cyclic reversible termination (CRT) is a promising method with the goal of producing accurate sequence information at a fraction of the cost and effort. The foundation of this approach is the reversible terminator (RT), its chemical and biological properties of which directly impact the performance of the sequencing technology. Here, we have discovered a novel paradigm in RT chemistry, the attachment of a photocleavable, 2-nitrobenzyl group to the N(6)-position of 2'-deoxyadenosine triphosphate (dATP), which, upon incorporation, terminates DNA synthesis. The 3'-OH group of the N(6)-(2-nitrobenzyl)-dATP remains unblocked, providing favorable incorporation and termination properties for several commercially available DNA polymerases while maintaining good discrimination against mismatch incorporations. Upon removal of the 2-nitrobenzyl group with UV light, the natural nucleotide is restored without molecular scarring. A five-base experiment, illustrating the exquisite, stepwise addition through a homopolymer repeat, demonstrates the applicability of the N(6)-(2-nitrobenzyl)-dATP as an ideal RT for CRT sequencing.

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.

Nucleoside and nucleobase antimetabolites have substantially impacted treatment of cancer and infections. Their close resemblance to natural analogs gives them the power to interfere with a variety of intracellular targets, which on one hand gives them high potency, but on the other hand incurs severe side effects, especially of the chemotherapeutics used against malignancies. Therefore, the development of novel nucleoside analogs with widened therapeutic windows represents an attractive target to synthetic organic and medicinal chemists. This review discusses the current antimetabolite drugs: 5- fluorouracil, 6-mercaptopurine, 6-thioguanine, Cladribine, Vidaza, Decitabine, Emtricitabine, Abacavir, Sorivudine, Clofarabine, Fludarabine, and Nelarabine; gives insight into the nucleoside drug candidates that are being developed; and outlines the approaches to nucleobase modifications that may help discover novel bioactive nucleoside analogs with the mechanism of action focused on termination of DNA synthesis, which is expected to diminish the off-target toxicity in non-proliferating human cells.

The thermolysis of C 60 H 2 to yield C 60 and H 2 was studied by hybrid density functional theory (B3LYP/6-311G**//B3LYP/3-21G). The concerted loss of dihydrogen requires an activation energy of 92 kcal mol −1 at T =452 K. An alternative radical mechanism, which is first order in the C 60 H 2 concentration, has an activation energy at 452 K of only 61 kcal mol −1 . Monitoring of the C 60 H 2 decomposition in 1,2-dichloro-[D 4 ]-benzene solution by NMR spectroscopy indicates a pseudo first-order reaction with an activation energy of 61.38±2.35 kcal mol −1 .

Calpains are a family of intracellular, calcium-dependent cysteine proteases involved in a variety of regulatory processes, including cytoskeletal dynamics, cell-cycle progression, signal transduction, gene expression, and apoptosis. These enzymes have been implicated in a number of disease processes, notably for this review involving eosinophilic tissue inflammation, such as eosinophilic esophagitis (EoE), a chronic inflammatory disorder triggered by allergic hypersensitivity to food and associated with genetic variants in calpain 14 (CAPN14). Herein we review the genetic, structural, and biochemical properties of CAPN14 and its gene product CAPN14, and its emerging role in patients with EoE. The CAPN14 gene is localized at chromosome 2p23.1-p21 and is most homologous to CAPN13 (36% sequence identity), which is located 365 kb downstream of CAPN14. Structurally, CAPN14 has classical calpain motifs, including a cysteine protease core. In comparison with other human calpains, CAPN14 has a unique expression pattern, with the highest levels in the upper gastrointestinal tract, particularly in the squamous epithelium of the esophagus. The CAPN14 gene is positioned in an epigenetic hotspot regulated by IL-13, a TH2 cytokine with increased levels in patients with EoE that has been shown to be a mediator of the disease. CAPN14 induces disruptive effects on the esophageal epithelium by impairing epithelial barrier function in association with loss of desmoglein-1 expression and has a regulatory role in repairing epithelial changes induced by IL-13. Thus CAPN14 is a unique protease with distinct tissue-specific expression and function in patients with EoE and is a potential therapeutic target for EoE and related eosinophilic and allergic diseases.

Current FDA-approved chemotherapeutic antimetabolites elicit severe side effects that warrant their improvement; therefore, we designed compounds with mechanisms of action focusing on inhibiting DNA replication rather than targeting multiple pathways. We previously discovered that 5-(α-substituted-2-nitrobenzyloxy)methyluridine-5′-triphosphates were exquisite DNA synthesis terminators; therefore, we synthesized a library of 35 thymidine analogs and evaluated their activity using an MTT cell viability assay of MCF7 breast cancer cells chosen for their vulnerability to these nucleoside derivatives. Compound 3a, having an α-tert-butyl-2-nitro-4-(phenyl)alkynylbenzyloxy group, showed an IC50 of 9 ± 1 μM. The compound is more selective for cancer cells than for fibroblast cells compared with 5-fluorouracil. Treatment of MCF7 cells with 3a elicits the DNA damage response as indicated by phosphorylation of γ-H2A. A primer extension assay of the 5′-triphosphate of 3a revealed that 3aTP is more likely to inhibit DNA polymerase than to lead to termination events upon incorporation into the DNA replication fork.

1-Chloro-3-methylenecyclopropene was prepared by vacuum gas-solid elimination of 2,2-dichloro-1-methylene-3-(trimethylsilyl)cyclopropane over solid cesium fluoride and characterized by 1 H and 13 C NMR spectroscopy at −103 °C.

The high resolution infrared spectrum of DCCN in the region of 2385–2530 cm−1 has been investigated using infrared kinetic spectroscopy. The CD stretching fundamental, ν1,together with the closely related hot bands, ν1+ν5−ν5,ν1+2ν5±2−2ν5±2, and ν1+2ν50−2ν50 have been observed and assigned. In addition the combination band, ν1+ν5, was observed. When the information provided by this perpendicular band is combined with that provided by ν1+ν5−ν5, the vibrational energy of ν5 is accurately determined to be 74.845(2) cm−1. This measurement, when combined with the ab initio and semirigid bender calculations of Malmquist et al., gives a barrier to linearity of 280 cm−1. Generally, fine structure splittings are not resolved for any levels except those belonging to the upper l- or K-type doubling components of ν1+ν5. This fine structure splitting is most strongly exhibited in the Q-branch of the ν1+ν5 band where it is 0.039 cm−1.

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.

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.

The molecular orientation of trimethylamine N-oxide (TMAO), a powerful protein stabilizer, was explored at aqueous/hydrophobic interfaces using vibrational sum frequency spectroscopy (VSFS). The systems studied included the octadecyltrichlorosilane (OTS)/water interface, which represents an aqueous solution in direct contact with a hydrophobic medium. Surprisingly, the measurements revealed that the methyl groups of TMAO pointed into the aqueous phase and away from the OTS. This orientation may arise from the more hydrophilic nature of methyl groups attached to a strongly electron-withdrawing atom such as a quaternary nitrogen. Additional studies were performed at the air/water interface. This interface showed a high degree of TMAO alignment, but the dangling OH from water was present even at 5 M TAMO. Moreover, the addition of this osmolyte modestly increased the surface tension of the interface. This meant that this species was somewhat depleted at the interface compared to the bulk solution. These findings may have implications for the stabilizing effect of TMAO on proteins. Specifically, the strong hydration required for the methyl groups as well as the oxide moiety should be responsible for the osmolyte's depletion from hydrophobic/aqueous interfaces. Such depletion effects should help stabilize proteins in their folded and native conformations on entropic grounds, although orientational effects may play an additional role.

1-Chloro-3-methylenecyclopropene was prepared by vacuum gas-solid elimination of 2,2-dichloro-1-methylene-3-(trimethylsilyl)cyclopropane over solid cesium fluoride and characterized by 1 H and 13 C NMR spectroscopy at −103 °C.

1,4-Dichlorospiropentadiene was prepared by vacuum gas-solid elimination of compound 3 over solid (n-Bu) 4 N + F − and characterized by 1 H and 13 C NMR spectroscopy at −103 °C.

The direct binding mechanism for urea-based denaturation of proteins was tested with a thermoresponsive polymer, poly(N-isopropylacrylamide) (PNIPAM). Thermodynamic measurements of the polymer's hydrophobic collapse were complemented by Fourier transform infrared (FTIR) spectroscopy, Stokes radius measurements, and methylated urea experiments. It was found that the lower critical solution temperature (LCST) of PNIPAM decreased as urea was added to the solution. Therefore, urea actually facilitated the hydrophobic collapse of the macromolecule. Moreover, these thermodynamic measurements were strongly correlated with amide I band data which indicated that the decrease in the LCST was coupled to the direct hydrogen bonding of urea to the amide moieties of the polymer. In addition, the hydrogen bonding was found to be highly cooperative, which is consistent with a cross-linking (bivalent binding) mechanism. Cross-linking was confirmed by Stokes radius measurements below the polymer's LCST using gel filtration chromatography. Finally, phase transition measurements with methylurea, dimethylurea, and tetramethylurea indicated that these substituted compounds caused the LCST of PNIPAM to rise with increasing methyl group content. No evidence could be found for the direct binding of any of these methylated ureas to the polymer amide moieties by FTIR. These results are inconsistent with a direct hydrogen-bonding mechanism for the urea-induced denaturation of proteins.

Background: Current chemotherapeutic antimetabolites often exhibit severe side effects that limit their use as drugs; therefore, we designed nucleoside compounds with mechanisms of action focusing on inhibiting

In breast tumors, activation of the nuclear factor κB (NFκB) and cyclooxygenase (COX)-prostaglandin pathways promote cell survival, migration, invasion, angiogenesis, and resistance to therapy - all phenotypes of aggressive disease where therapy options remain limited. Thus, adding an anti-inflammatory agent to breast cancer treatment would be beneficial but no such drugs have been approved, either as mono- or adjuvant therapy. Moreover, redundancy and interdependence between inflammatory signaling implies that blocking one pathway is unlikely to be effective. We rationalized that targeting of multiple inflammatory pathways can be achieved via a hybrid drug, which, by definition, is a chemical entity capable of interacting simultaneously with multiple targets. Hence, hybrid drugs consisting of clinically relevant moieties aimed at multiple inflammatory pathways were synthesized and tested. To target the NFκB pathway, we utilized dimethyl fumarate (DMF), a safe and effective anti-inflammatory drug already in clinical use. For the first time, we show that DMF effectively blocks NFκB activity in breast cancer cells. To target the COX2-PGE2 axis in breast cancer cells, we selected the classical NSAID aspirin (ASA), given its beneficial effect in reducing breast cancer incidence with extended ASA use. Our first hybrid drug, GTCpFE, consists of DMF linked to ASA. We find that GTCpFE inhibits NFκB activity as does DMF, while retaining its ASA-like activity. GTCpFE blocks p65 nuclear entry like DMF but, in addition, also inhibits key kinases (IKKα/β) in the NFκB pathway. This suggests that GTCpFE as a hybrid drug has gained new activities over parental DMF or ASA. GTCpFE inhibits breast cancer stem cells, an important NFκB- and PGE2-dependent phenotype in aggressive cancers, more effectively than ASA, DMF, or the combination of the two. Thus, as a proof-of-principle, the hybrid drug GTCpFE shows increased efficacy and potency by modulating multiple inflammatory pathways. Because an anti-inflammatory therapy is not cytocidal on its own on the bulk of the tumor, we decided to combine DMF with more conventional therapies. Given that majority of breast cancers express estrogen receptor (ER), our second hybrid drug, Ral-Fum, consists of DMF linked to Raloxifene (Ral), a selective ER-modulator, to specifically target ER+ breast cancers. This hybrid drug shows improved anti-NFκB activity over DMF while maintaining ER antagonism, hence is both an endocrine therapy and an anti-inflammatory agent. Moreover, Ral-Fum attenuates the formation of endocrine resistant colonies. Taken together, our results indicate that a hybrid drug strategy to target essential factors for breast cancer cell proliferation and survival, such as ER, and multiple aspects of inflammation can generate promising and novel therapeutic agents to treat aggressive breast cancers. Citation Format: Irida Kastrati, Marton Siklos, Vladislav Litosh, Gregory Thatcher, Jonna Frasor. Design and synthesis of hybrid drugs to target inflammatory pathways in breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4497. doi:10.1158/1538-7445.AM2015-4497

Thermolysis of spiro[2.4]hepta-1,4,6-triene (1a) at 50 degrees C yielded bicyclo[3.2.0]hepta-1,3,6-triene (5), which dimerized in two different fashions to form cyclobutanes. The 1,2-dimethyl and 1-propyl derivatives of 1a also rearranged at 50 degrees C, but at a faster rate, each yielding a pair of cyclobutane dimers. The structures of these symmetrical dimers were investigated by 1D and 2D NMR and NOE difference spectroscopy. Ab initio calculations indicated that the two strained olefins 1a and 5 had comparable energies about 50 kcal/mol lower than norborna-1(7),2,5-triene, which was thus excluded as a reaction intermediate.

[figure: see text] Benzocyclopropene and cyclopropa[b]naphthalene react with dichloro-bis(tricyclohexylphosphine)methylideneruthenium, incorporating the metallacarbene to form unstable 3-ruthenacyclopentenes, which decompose to give o-xylylenes that can be trapped as Diels-Alder adducts by dimethyl acetylenedicarboxylate. In contrast, bis(eta 5-cyclopentadienyl)methylidenetitanium forms moderately stable 2- and 3-titanacyclopentene complexes.

Approximately 75% of breast cancers express estrogen receptor (ER), classified as either ER+ luminal A or luminal B. Five year survival rate of luminal B breast cancers is ∼50% compared to ∼95% for luminal A, making luminal B subtype a target for better understanding the mechanisms of carcinogenicity and improved therapy. Inflammation and activation of its nuclear factor NFkB, is linked clinically and epidemiologically to cancer. Activation of NFkB in a subset of ER+ cancers is associated with poor prognosis and resistance to therapy. We were the first to show synergy between ER and NFkB in a specific gene signature that highly correlates with luminal B ER+ breast cancers, suggesting a potential functional role for NFkB inhibition in this subset. Based on these findings, we hypothesized that co-targeting ER and NFkB may achieve clinical efficacy in luminal B breast cancer. To target the ER, a selective estrogen receptor modulator (SERM) was employed. To target NFkB pathway, an NSAID-based therapy would be safe and, based on epidemiological data, is suggested to be chemopreventive in breast cancer. For NFkB targeting, we characterized a family of aspirin (ASA)-based analogs (x-ASA) that also serve as pro-drugs for quinone methide formation. We found that x-ASA analogs inhibited TNFa induction of NFkB via a reporter assay in a dose-dependent fashion in MCF-7 cells, and also shut down several genes typically up-regulated by the ER-NFkB crosstalk. Next we tested the co-targeting approach in mammosphere formation assays: the combination significantly reduced overall mammosphere number and size. While the mechanisms of action remains to be elucidated, the aspirin analogs in combination with a SERM show promise in treating ER+ breast cancers with activated NFkB where standard endocrine therapy alone fails. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3732. doi:1538-7445.AM2012-3732

We describe a novel 3'-OH unblocked reversible terminator with the potential to improve accuracy and read-lengths in next-generation sequencing (NGS) technologies. This terminator is based on 5-hydroxymethyl-2'-deoxyuridine triphosphate (HOMedUTP), a hypermodified nucleotide found naturally in the genomes of numerous bacteriophages and lower eukaryotes. A series of 5-(2-nitrobenzyloxy)methyl-dUTP analogs (dU.I-dU.V) were synthesized based on our previous work with photochemically cleavable terminators. These 2-nitrobenzyl alkylated HOMedUTP analogs were characterized with respect to incorporation, single-base termination, nucleotide selectivity and photochemical cleavage properties. Substitution at the α-methylene carbon of 2-nitrobenzyl with alkyl groups of increasing size was discovered as a key structural feature that provided for the molecular tuning of enzymatic properties such as single-base termination and improved nucleotide selectivity over that of natural nucleotides. 5-[(S)-α-tert-Butyl-2-nitrobenzyloxy]methyl-dUTP (dU.V) was identified as an efficient reversible terminator, whereby, sequencing feasibility was demonstrated in a cyclic reversible termination (CRT) experiment using a homopolymer repeat of ten complementary template bases without detectable UV damage during photochemical cleavage steps. These results validate our overall strategy of creating 3'-OH unblocked reversible terminator reagents that, upon photochemical cleavage, transform back into a natural state. Modified nucleotides based on 5-hydroxymethyl-pyrimidines and 7-deaza-7-hydroxymethyl-purines lay the foundation for development of a complete set of four reversible terminators for application in NGS technologies.

1,4-Dichlorospiropentadiene was prepared by vacuum gas-solid elimination of compound 3 over solid (n-Bu) 4 N + F − and characterized by 1 H and 13 C NMR spectroscopy at −103 °C.

Introduction Activation of cyclooxygenase (COX)/prostaglandin and nuclear factor κB (NFκB) pathways can promote breast tumor initiation, growth, and progression to drug resistance and metastasis. Thus, anti-inflammatory drugs have been widely explored as chemopreventive and antineoplastic agents. Aspirin (ASA), in particular, is associated with reduced breast cancer incidence but gastrointestinal toxicity has limited its usefulness. To improve potency and minimize toxicity, ASA ester prodrugs have been developed, in which the carboxylic acid of ASA is masked and ancillary pharmacophores can be incorporated. To date, the effects of ASA and ASA prodrugs have been largely attributed to COX inhibition and reduced prostaglandin production. However, ASA has also been reported to inhibit the NFκB pathway at very high doses. Whether ASA prodrugs can inhibit NFκB signaling remains relatively unexplored. Methods A library of ASA prodrugs was synthesized and screened for inhibition of NFκB activity and cancer stem-like cell (CSC) properties, an important PGE2-and NFκB-dependent phenotype of aggressive breast cancers. Inhibition of NFκB activity was determined by dual luciferase assay, RT-QPCR, p65 DNA binding activity and Western blots. Inhibition of CSC properties was determined by mammosphere growth, CD44+CD24−immunophenotype and tumorigenicity at limiting dilution. Results While we identified multiple ASA prodrugs that are capable of inhibiting the NFκB pathway, several were associated with cytotoxicity. Of particular interest was GTCpFE, an ASA prodrug with fumarate as the ancillary pharmacophore. This prodrug potently inhibits NFκB activity without innate cytotoxicity. In addition, GTCpFE exhibited selective anti-CSC activity by reducing mammosphere growth and the CD44+CD24−immunophenotype. Moreover, GTCpFE pre-treated cells were less tumorigenic and, when tumors did form, latency was increased and growth rate was reduced. Structure-activity relationships for GTCpFE indicate that fumarate, within the context of an ASA prodrug, is essential for anti-NFκB activity, whereas both the ASA and fumarate moieties contributed to attenuated mammosphere growth. Conclusions These results establish GTCpFE as a prototype for novel ASA-and fumarate-based anti-inflammatory drugs that: (i) are capable of targeting CSCs, and (ii) may be developed as chemopreventive or therapeutic agents in breast cancer. Electronic supplementary material The online version of this article (doi:10.1186/s12885-015-1868-7) contains supplementary material, which is available to authorized users.

NO/cGMP signaling is essential for normal brain function, including learning and memory, and mediation of long-term potentiation (LTP). NO/cGMP signaling is coupled to cholinergic, glutamatergic, and dopaminergic systems and plays key roles in motor function associated with Parkinson’s disease pathogenesis and L-DOPA therapy. In Alzheimer’s Disease (AD), early synaptic failure, has been linked to dysfunction of gene expression programs mediated via the transcription factor cAMP-response element binding protein (CREB), activation of which is tightly regulated by NO/cGMP. Since several neurodegenerative disorders continue to be dogmatically linked to the chemical toxicity of NO, activation of soluble guanylyl cyclase (sGC) represents a potential therapeutic approach in both AD and PD; whereas negative modulation of sGC may be of use in some stages of PD. Such small molecule sGC modulators (GCMs) can be assayed in cell cultures by measuring levels of phosphorylated CREB (pCREB). GCMs that allosterically potentiate NO activation of sGC have proven successful in clinical trials for peripheral indications, however, there are no reports directed at therapeutic activity in the CNS. The aim of this study is to develop GCMs for use in CNS disorders, including AD and PD. A library of GCMs was synthesized, including positive allosteric modulators (PAMs) and negative modulators, as assessed by increasing levels of pCREB in SH-SY5Y human neuroblastoma cell cultures. Molecules were designed by classical bioisosteric replacement, aiming for desirable physiochemical properties and chemical diversity. GCM PAMs were assayed for cGMP elevation and reversal of neurotoxicity induced by 6-hydroxydopamine in dopaminergic neuronal cells. One GCM PAM, active in cell culture, was further tested for the reversal of memory deficits in mice treated with scopolamine, and drug levels in brain and plasma measured. The ligand binding site for sGC stimulators and GCM PAMs on the sGC protein remains to be definitively defined. Photoaffinity probes were designed that retained the activity of the parent GCM PAM in cell cultures in order to elucidate the binding mode to sGC and to indicate other protein partners for such compounds.