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1.
Bioorg Med Chem Lett ; 94: 129432, 2023 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-37591319

RESUMO

Nucleoside and nucleobase analogs capable of interfering with nucleic acid synthesis have played essential roles in fighting infectious diseases. However, many of these agents are associated with important and potentially lethal off-target intracellular effects that limit their use. Based on the previous discovery of base-modified 2'-deoxyuridines, which showed high anticancer activity while exhibiting lower toxicity toward rapidly dividing normal human cells compared to antimetabolite chemotherapeutics, we hypothesized that a similar modification of the N4-hydroxycytidine (NHC) molecule would provide novel antiviral compounds with diminished side effects. This presumption is due to the substantial structural difference with natural cytidine leading to less recognizability by host cell enzymes. Among the 42 antimetabolite species that have been synthesized and screened against VEEV, one hit compound was identified. The structural features of the modifying moiety were similar to those of the anticancer lead 2'-deoxyuridine derivative reported previously, providing an opportunity to pursue further structure-activity relationship (SAR) studies directed to lead improvement, and obtain insight into the mechanism of action, which can lead to identifying drug candidates against a broad spectrum of RNA viral infections.


Assuntos
Vírus da Encefalite Equina Venezuelana , Animais , Humanos , Antimetabólitos , Antivirais/farmacologia , Desoxiuridina , Cavalos , Imunossupressores
2.
Anal Biochem ; 622: 114116, 2021 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-33716126

RESUMO

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.


Assuntos
Aldose-Cetose Isomerases/análise , Dicroísmo Circular/métodos , Ensaios Enzimáticos/métodos , Proteínas de Bactérias/metabolismo , Catálise , Francisella tularensis/metabolismo , Lipopolissacarídeos/metabolismo , Pentosefosfatos/metabolismo , Ribulosefosfatos/análise , Ribulosefosfatos/metabolismo , Especificidade por Substrato , Açúcares Ácidos/metabolismo , Fosfatos Açúcares/metabolismo
3.
Cephalalgia ; 38(8): 1471-1484, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29022756

RESUMO

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.


Assuntos
Hiperalgesia/induzido quimicamente , Hiperalgesia/enzimologia , Transtornos de Enxaqueca/enzimologia , Guanilil Ciclase Solúvel/fisiologia , Antagonistas Adrenérgicos beta/administração & dosagem , Antagonistas Adrenérgicos beta/uso terapêutico , Regulação Alostérica , Animais , Anticonvulsivantes/administração & dosagem , Anticonvulsivantes/uso terapêutico , Peptídeo Relacionado com Gene de Calcitonina/biossíntese , Modelos Animais de Doenças , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/uso terapêutico , Feminino , Hiperalgesia/tratamento farmacológico , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Transtornos de Enxaqueca/induzido quimicamente , Transtornos de Enxaqueca/tratamento farmacológico , Transtornos de Enxaqueca/etiologia , Terapia de Alvo Molecular , Óxido Nítrico/efeitos adversos , Óxido Nítrico/metabolismo , Doadores de Óxido Nítrico/farmacologia , Nitroglicerina/farmacologia , Oxidiazóis/administração & dosagem , Oxidiazóis/metabolismo , Oxidiazóis/uso terapêutico , Propranolol/administração & dosagem , Propranolol/uso terapêutico , Proteínas Proto-Oncogênicas c-fos/biossíntese , Quinoxalinas/administração & dosagem , Quinoxalinas/metabolismo , Quinoxalinas/uso terapêutico , Agonistas do Receptor 5-HT1 de Serotonina/administração & dosagem , Agonistas do Receptor 5-HT1 de Serotonina/uso terapêutico , Guanilil Ciclase Solúvel/metabolismo , Sumatriptana/administração & dosagem , Sumatriptana/uso terapêutico , Topiramato/administração & dosagem , Topiramato/uso terapêutico
4.
J Allergy Clin Immunol ; 139(6): 1762-1771.e7, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28131390

RESUMO

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.


Assuntos
Calpaína/genética , Esofagite Eosinofílica/genética , Animais , Calpaína/química , Humanos , Conformação Proteica
5.
Chem Res Toxicol ; 29(7): 1151-9, 2016 07 18.
Artigo em Inglês | MEDLINE | ID: mdl-27258437

RESUMO

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 chemopreventive 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.


Assuntos
NF-kappa B/metabolismo , Pró-Fármacos/farmacocinética , Quinonas/farmacocinética , Ativação Metabólica , Células HT29 , Humanos , Espectrometria de Massas , Fator 2 Relacionado a NF-E2/metabolismo , Proteômica , Teoria Quântica
6.
JCI Insight ; 1(4): e86355, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-27158675

RESUMO

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.

7.
J Alzheimers Dis ; 49(3): 707-21, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26484927

RESUMO

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-ß, 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-ß 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.


Assuntos
Doença de Alzheimer/tratamento farmacológico , Glicoproteínas/uso terapêutico , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Animais , Inibidores de Cisteína Proteinase/farmacologia , Inibidores de Cisteína Proteinase/uso terapêutico , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Medo/efeitos dos fármacos , Glicoproteínas/química , Glicoproteínas/farmacologia , Hipocampo/citologia , Humanos , Técnicas In Vitro , Potenciação de Longa Duração/efeitos dos fármacos , Potenciação de Longa Duração/genética , Aprendizagem em Labirinto/efeitos dos fármacos , Memória/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos ICR , Camundongos Transgênicos , Mutação/genética , Técnicas de Patch-Clamp , Fragmentos de Peptídeos/metabolismo , Presenilina-1/genética , Espectrina/metabolismo
8.
Curr Top Med Chem ; 16(11): 1231-41, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26369814

RESUMO

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.


Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Nucleosídeos/química , Nucleosídeos/farmacologia , Proliferação de Células/efeitos dos fármacos , Humanos , Estrutura Molecular , Neoplasias/tratamento farmacológico , Neoplasias/patologia
9.
BMC Cancer ; 15: 845, 2015 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-26530254

RESUMO

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.


Assuntos
Aspirina/administração & dosagem , Neoplasias da Mama/tratamento farmacológico , NF-kappa B/genética , Pró-Fármacos/administração & dosagem , Aspirina/síntese química , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Feminino , Fumaratos/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Células MCF-7 , NF-kappa B/antagonistas & inibidores , NF-kappa B/biossíntese , Células-Tronco Neoplásicas/efeitos dos fármacos , Pró-Fármacos/síntese química , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
10.
Bioorg Med Chem ; 23(8): 1869-81, 2015 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-25778768

RESUMO

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±1µM. 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.


Assuntos
Antimetabólitos Antineoplásicos/química , Antimetabólitos Antineoplásicos/farmacologia , Replicação do DNA/efeitos dos fármacos , Inibidores da Síntese de Ácido Nucleico/química , Inibidores da Síntese de Ácido Nucleico/farmacologia , Timidina/análogos & derivados , Timidina/farmacologia , Neoplasias da Mama/tratamento farmacológico , Feminino , Humanos , Células MCF-7
11.
J Med Chem ; 56(15): 6054-68, 2013 Aug 08.
Artigo em Inglês | MEDLINE | ID: mdl-23834438

RESUMO

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.


Assuntos
Calpaína/antagonistas & inibidores , Compostos de Epóxi/síntese química , Leucina/análogos & derivados , Peptidomiméticos/síntese química , Calpaína/química , Domínio Catalítico , Química Click , Simulação por Computador , Desenho de Fármacos , Compostos de Epóxi/química , Cinética , Leucina/síntese química , Leucina/química , Simulação de Acoplamento Molecular , Papaína/antagonistas & inibidores , Peptidomiméticos/química , Estereoisomerismo , Relação Estrutura-Atividade
12.
Chem Res Toxicol ; 25(12): 2725-36, 2012 Dec 17.
Artigo em Inglês | MEDLINE | ID: mdl-23035985

RESUMO

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.


Assuntos
Aspirina/análogos & derivados , Aspirina/farmacologia , Indolquinonas/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Pró-Fármacos/farmacologia , Animais , Linhagem Celular Tumoral , Dano ao DNA , Glutationa/metabolismo , Glutationa S-Transferase pi/metabolismo , Humanos , Proteína 1 Associada a ECH Semelhante a Kelch , Camundongos , NAD(P)H Desidrogenase (Quinona)/metabolismo , Óxido Nítrico/metabolismo
14.
J Am Chem Soc ; 133(46): 18707-12, 2011 Nov 23.
Artigo em Inglês | MEDLINE | ID: mdl-21967088

RESUMO

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.


Assuntos
Metilaminas/química , Ar , Interações Hidrofóbicas e Hidrofílicas , Espectrometria de Massas , Proteínas/química , Água/química
15.
ACS Chem Neurosci ; 2(5): 256-268, 2011 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-21731800

RESUMO

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 ERß 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.

16.
Nucleic Acids Res ; 39(6): e39, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21227920

RESUMO

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.


Assuntos
Nucleotídeos de Desoxiuracil/química , Análise de Sequência de DNA/métodos , DNA Polimerase Dirigida por DNA/metabolismo , Nucleotídeos de Desoxiuracil/síntese química , Nucleotídeos de Desoxiuracil/metabolismo , Processos Fotoquímicos , Reação em Cadeia da Polimerase , Moldes Genéticos , Raios Ultravioleta
17.
J Am Chem Soc ; 131(26): 9304-10, 2009 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-19527028

RESUMO

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.


Assuntos
Acrilamidas/química , Oligopeptídeos/química , Polímeros/química , Proteínas/química , Ureia/química , Acrilamidas/análise , Resinas Acrílicas , Sítios de Ligação , Cromatografia em Gel , Escherichia coli/genética , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Oligopeptídeos/análise , Oligopeptídeos/genética , Oligopeptídeos/isolamento & purificação , Polímeros/análise , Desnaturação Proteica , Proteínas/análise , Espectroscopia de Infravermelho com Transformada de Fourier , Termodinâmica , Temperatura de Transição , Ureia/análogos & derivados
18.
Nucleic Acids Res ; 35(19): 6339-49, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17881370

RESUMO

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.


Assuntos
DNA Polimerase Dirigida por DNA/metabolismo , DNA/biossíntese , Nucleotídeos de Desoxiadenina/química , Análise de Sequência de DNA/métodos , Alquilação , Pareamento Incorreto de Bases , Nucleotídeos de Desoxiadenina/síntese química , Nucleotídeos de Desoxiadenina/efeitos da radiação , Desoxiadenosinas/síntese química , Desoxiadenosinas/química , Fotoquímica , Raios Ultravioleta
19.
J Org Chem ; 67(13): 4436-40, 2002 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-12076139

RESUMO

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.

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