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1.
Eur J Med Chem ; 227: 113926, 2022 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-34735919

RESUMO

Polo-like kinase 1 (PLK1) is a serine/threonine-protein kinase involved in cell cycle regulation and mitotic progression. Studies have shown that PLK1 is upregulated in many tumors and high levels are adversely related to a poor prognosis. Knocking down or inhibiting PLK1 results in synthetic lethality in PTEN deficient prostate tumors and Kras mutant colorectal tumors, further validating PLK1 as an oncotarget. Substrate recognition by PLK1 occurs through the Polo-Box Domain (PBD), which is a phospho-peptide binding site also responsible for subcellular localization. Much effort has been directed to target this kinase therapeutically through the ATP-binding site, and a few such inhibitors have advanced to clinical trials however with limited clinical efficacy. Moreover, it has been shown that a point mutation in PLK1 (C67V) confers dramatic cellular resistance to catalytic site inhibitors. An alternative approach to target PLK1 potently and selectively is through the PBD to block its protein-protein interactions. Through the REPLACE strategy, for converting peptide inhibitors into more drug-like non peptidic compounds, a PBD targeting compound series ("ABBAs"), has been identified and the key determinants of potency and selectivity elucidated through structure-activity relationship studies. In cellular experiments, the ABBAs were shown to lead to profound effects on the cell cycle, to inhibit tumor proliferation and overcome resistance of cells expressing the PLK1 C67V mutant to ATP-based inhibitors. These non-ATP competitive inhibitors of PLK1 were also used chemical biology probes to investigate the gene regulatory effects of PLK1, known to act on transcription factors such as p53.


Assuntos
Trifosfato de Adenosina/farmacologia , Antineoplásicos/farmacologia , Proteínas de Ciclo Celular/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Trifosfato de Adenosina/síntese química , Trifosfato de Adenosina/química , Antineoplásicos/síntese química , Antineoplásicos/química , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Ligantes , Estrutura Molecular , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Relação Estrutura-Atividade , Quinase 1 Polo-Like
2.
Eur J Med Chem ; 213: 113200, 2021 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-33524686

RESUMO

The rise in multidrug-resistant bacteria defines the need for identification of new antibacterial agents that are less prone to resistance acquisition. Compounds that simultaneously inhibit multiple bacterial targets are more likely to suppress the evolution of target-based resistance than monotargeting compounds. The structurally similar ATP binding sites of DNA gyrase and topoisomerase Ⅳ offer an opportunity to accomplish this goal. Here we present the design and structure-activity relationship analysis of balanced, low nanomolar inhibitors of bacterial DNA gyrase and topoisomerase IV that show potent antibacterial activities against the ESKAPE pathogens. For inhibitor 31c, a crystal structure in complex with Staphylococcus aureus DNA gyrase B was obtained that confirms the mode of action of these compounds. The best inhibitor, 31h, does not show any in vitro cytotoxicity and has excellent potency against Gram-positive (MICs: range, 0.0078-0.0625 µg/mL) and Gram-negative pathogens (MICs: range, 1-2 µg/mL). Furthermore, 31h inhibits GyrB mutants that can develop resistance to other drugs. Based on these data, we expect that structural derivatives of 31h will represent a step toward clinically efficacious multitargeting antimicrobials that are not impacted by existing antimicrobial resistance.


Assuntos
Trifosfato de Adenosina/farmacologia , Antibacterianos/farmacologia , DNA Girase/metabolismo , DNA Topoisomerase IV/antagonistas & inibidores , Escherichia coli/efeitos dos fármacos , Staphylococcus aureus/efeitos dos fármacos , Trifosfato de Adenosina/síntese química , Trifosfato de Adenosina/química , Antibacterianos/síntese química , Antibacterianos/química , Cristalografia por Raios X , DNA Topoisomerase IV/metabolismo , Relação Dose-Resposta a Droga , Escherichia coli/enzimologia , Escherichia coli/patogenicidade , Testes de Sensibilidade Microbiana , Simulação de Acoplamento Molecular , Estrutura Molecular , Staphylococcus aureus/enzimologia , Staphylococcus aureus/patogenicidade , Relação Estrutura-Atividade
3.
Molecules ; 25(23)2020 Nov 24.
Artigo em Inglês | MEDLINE | ID: mdl-33255222

RESUMO

RNA 5'-modifications are known to extend the functional spectrum of ribonucleotides. In recent years, numerous non-canonical 5'-modifications, including adenosine-containing cofactors from the group of B vitamins, have been confirmed in all kingdoms of life. The structural component of thiamine adenosine triphosphate (thiamine-ATP), a vitamin B1 derivative found to accumulate in Escherichia coli and other organisms in response to metabolic stress conditions, suggests an analogous function as a 5'-modification of RNA. Here, we report the synthesis of thiamine adenosine dinucleotides and the preparation of pure 5'-thiamine-capped RNAs based on phosphorimidazolide chemistry. Furthermore, we present the incorporation of thiamine-ATP and thiamine adenosine diphosphate (thiamine-ADP) as 5'-caps of RNA by T7 RNA polymerase. Transcripts containing the thiamine modification were modified specifically with biotin via a combination of thiazole ring opening, nucleophilic substitution and copper-catalyzed azide-alkyne cycloaddition. The highlighted methods provide easy access to 5'-thiamine RNA, which may be applied in the development of thiamine-specific RNA capture protocols as well as the discovery and confirmation of 5'-thiamine-capped RNAs in various organisms.


Assuntos
Técnicas de Química Sintética , Capuzes de RNA/química , RNA/síntese química , Tiamina/química , Trifosfato de Adenosina/síntese química , Trifosfato de Adenosina/química , Biotinilação , Catálise , RNA Polimerases Dirigidas por DNA , Estrutura Molecular , RNA/química , RNA/genética , Tiamina Trifosfato/síntese química , Tiamina Trifosfato/química , Proteínas Virais
4.
Cell Chem Biol ; 27(8): 1073-1083.e12, 2020 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-32521230

RESUMO

ATP is an important energy metabolite and allosteric signal in health and disease. ATP-interacting proteins, such as P2 receptors, control inflammation, cell death, migration, and wound healing. However, identification of allosteric ATP sites remains challenging, and our current inventory of ATP-controlled pathways is likely incomplete. Here, we develop and verify mipATP as a minimally invasive photoaffinity probe for ATP-interacting proteins. Its N6 functionalization allows target enrichment by UV crosslinking and conjugation to reporter tags by "click" chemistry. The additions are compact, allowing mipATP to completely retain the calcium signaling responses of native ATP in vitro and in vivo. mipATP specifically enriched for known nucleotide binders in A549 cell lysates and membrane fractions. In addition, it retrieved unannotated ATP interactors, such as the FAS receptor, CD44, and various SLC transporters. Thus, mipATP is a promising tool to identify allosteric ATP sites in the proteome.


Assuntos
Trifosfato de Adenosina/metabolismo , Membrana Celular/metabolismo , Proteoma/análise , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/síntese química , Aminoácidos/química , Aminoácidos/metabolismo , Animais , Animais Geneticamente Modificados/metabolismo , Sinalização do Cálcio , Calmodulina/genética , Calmodulina/metabolismo , Linhagem Celular Tumoral , Membrana Celular/química , Cromatografia Líquida de Alta Pressão , Química Click , Corantes Fluorescentes/química , Humanos , Marcação por Isótopo , Larva/metabolismo , Imagem Óptica , Proteoma/metabolismo , Espectrometria de Massas em Tandem , Raios Ultravioleta , Peixe-Zebra/crescimento & desenvolvimento , Peixe-Zebra/metabolismo
5.
J Phys Chem Lett ; 10(15): 4192-4196, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31305079

RESUMO

We report the successful one-pot synthesis of adenosine mono-, di-, and triphosphate in the confined space of a mordenite zeolite. This is also the first report of ATP synthesized onto a porous mineral surface. The results revealed a plausible prebiotic route to ribonucleotides and highlighted the contribution of microporous minerals in the origins of life.


Assuntos
Difosfato de Adenosina/síntese química , Monofosfato de Adenosina/síntese química , Trifosfato de Adenosina/síntese química , Silicatos de Alumínio/química , Nanopartículas/química , Técnicas de Síntese em Fase Sólida/métodos , Zeolitas/química , Adenina/química , Organofosfatos/química , Origem da Vida , Porosidade , Ribose/química , Propriedades de Superfície , Termodinâmica , Fatores de Tempo
6.
Acc Chem Res ; 52(6): 1623-1631, 2019 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-30882207

RESUMO

Molecular machines are an important and emerging frontier in research encompassing interdisciplinary subjects of chemistry, physics, biology, and nanotechnology. Although there has been major interest in creating synthetic molecular machines, research on natural molecular machines is also crucial. Biomolecular motors are natural molecular machines existing in nearly every living systems. They play a vital role in almost every essential process ranging from intracellular transport to cell division, muscle contraction and the biosynthesis of ATP that fuels life processes. The construction of biomolecular motor-based biomimetic systems can help not only to deeply understand the mechanisms of motor proteins in the biological process but also to push forward the development of bionics and biomolecular motor-based devices or nanomachines. From combination of natural biomolecular motors with supramolecular chemistry, great opportunities could emerge toward the development of intelligent molecular machines and biodevices. In this Account, we describe our efforts to design and reconstitute biomolecular motor-based active biomimetic systems, in particular, the combination of motor proteins with layer-by-layer (LbL) assembled cellular structures. They are divided into two parts: (i) reconstitution of rotary molecular motor FOF1-ATPase, which is coated on the surface of LbL assembled microcapsules or multilayers and synthesizes adenosine triphosphate (ATP) through creating a proton gradient; (ii) molecular assembly of linear molecular motors, the kinesin-based active biomimetic systems, which are coated on a planar surface or LbL assembled tubular structure and drive the movement of microtubules. LbL assembled structures offer motor proteins with an environment that resembles the natural cell. This enables high activity and optimized function of the motor proteins. The assembled biomolecular motors can mimic their functionalities from the natural system. In addition, LbL assembly provides facile integration of functional components into motor protein-based active biomimetic systems and achieves the manipulation of FOF1-ATPase and kinesin. For FOF1-ATPase, the light-driven proton gradient and controlled ATP synthesis are highlighted. For kinesin, the strategies used for the direction and velocity control of kinesin-based molecular shuttles are discussed. We hope this research can inspire new ideas and propel the actual applications of biomolecular motor-based devices in the future.


Assuntos
Biomimética/métodos , Cinesinas/química , Membranas Artificiais , Complexo de Proteína do Fotossistema II/química , ATPases Translocadoras de Prótons/química , Trifosfato de Adenosina/síntese química , Nanotecnologia/métodos , Prótons
7.
Angew Chem Int Ed Engl ; 58(15): 4896-4900, 2019 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-30701643

RESUMO

Herein, we present a light-gated protocell model made of plasmonic colloidal capsules (CCs) assembled with bacteriorhodopsin for converting solar energy into electrochemical gradients to drive the synthesis of energy-storage molecules. This synthetic protocell incorporated an important intrinsic property of noble metal colloidal particles, namely, plasmonic resonance. In particular, the near-field coupling between adjacent metal nanoparticles gave rise to strongly localized electric fields and resulted in a broad absorption in the whole visible spectra, which in turn promoted the flux of photons to bacteriorhodopsin and accelerated the proton pumping kinetics. The cell-like potential of this design was further demonstrated by leveraging the outward pumped protons as "chemical signals" for triggering ATP biosynthesis in a coexistent synthetic protocell population. Hereby, we lay the ground work for the engineering of colloidal supraparticle-based synthetic protocells with higher-order functionalities.


Assuntos
Trifosfato de Adenosina/síntese química , Células Artificiais/química , Luz , Fótons , Ressonância de Plasmônio de Superfície , Trifosfato de Adenosina/química , Bacteriorodopsinas/química , Engenharia Celular , Concentração de Íons de Hidrogênio , Tamanho da Partícula , Propriedades de Superfície
8.
J Am Chem Soc ; 140(24): 7568-7578, 2018 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-29787268

RESUMO

Bisphosphonates are a major class of drugs used to treat osteoporosis, Paget's disease, and cancer. They have been proposed to act by inhibiting one or more targets including protein prenylation, the epidermal growth factor receptor, or the adenine nucleotide translocase. Inhibition of the latter is due to formation in cells of analogs of ATP: the isopentenyl ester of ATP (ApppI) or an AppXp-type analog of ATP, such as AMP-clodronate (AppCCl2p). We screened both ApppI as well as AppCCl2p against a panel of 369 kinases finding potent inhibition of some tyrosine kinases by AppCCl2p, attributable to formation of a strong hydrogen bond between tyrosine and the terminal phosphonate. We then synthesized bisphosphonate preprodrugs that are converted in cells to other ATP-analogs, finding low nM kinase inhibitors that inhibited cell signaling pathways. These results help clarify our understanding of the mechanisms of action of bisphosphonates, potentially opening up new routes to the development of bone resorption, anticancer, and anti-inflammatory drug leads.


Assuntos
Trifosfato de Adenosina/análogos & derivados , Difosfonatos/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais/efeitos dos fármacos , Trifosfato de Adenosina/síntese química , Trifosfato de Adenosina/química , Trifosfato de Adenosina/farmacologia , Linhagem Celular Tumoral , Difosfonatos/síntese química , Difosfonatos/química , Humanos , Ligação de Hidrogênio , Modelos Químicos , Pró-Fármacos/síntese química , Pró-Fármacos/química , Pró-Fármacos/farmacologia , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Proteínas Tirosina Quinases/antagonistas & inibidores
9.
Artigo em Inglês | MEDLINE | ID: mdl-28869872

RESUMO

An efficient high-performance countercurrent chromatography (HPCCC) based method has been developed for the purification of chemically synthesized 1-adenosin-5'-yl 3-(3-methylbut-3-enyl)triphosphoric acid diester (ApppI). ApppI is an adenosine triphosphate (ATP) analogue with biological significance due to its varied actions in the body. ApppI was synthesized and purified as its tetrabutylammonium (TBA) salt and converted successfully into its more practical sodium salt form after purification. The amount of TBA hydroxide (2.0, 2.5 and 3.0 eq) used in the synthesis of ApppI was shown to exert an effect on the purification process with HPCCC and on the overall yield (8%, 16% and 22%, respectively). 1-Adenosin-5'-yl 3-(3-methylbut-3-enyl)diphosphoric acid diester (AppI) was also isolated as a side product.


Assuntos
Trifosfato de Adenosina/análogos & derivados , Distribuição Contracorrente/métodos , Trifosfato de Adenosina/síntese química , Trifosfato de Adenosina/isolamento & purificação , Temperatura
10.
Bioorg Med Chem Lett ; 27(13): 2838-2848, 2017 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-28506751

RESUMO

The frequency of PI3K/Akt/mTOR (PAM) Pathway mutations in human cancers sparked interest to determine if the pathway is druggable. The modest clinical benefit observed with mTOR rapalogs (temsirolimus and everolimus) provided further motivation to identify additional nodes of pathway inhibition that lead to improved clinical benefit. Akt is a central signaling node of the PAM pathway and could be an ideal target for improved pathway inhibition. Furthermore, inhibitors of Akt may be especially beneficial in tumors with Akt1 mutations. Recently, multiple ATP-competitive Akt inhibitors have been identified and are currently in clinical development. This review details the medicinal chemistry efforts towards identification of these molecules, highlights relevant preclinical data supporting clinical evaluation, and summarizes current clinical development plans.


Assuntos
Trifosfato de Adenosina/farmacologia , Piperazinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Pirimidinas/farmacologia , Trifosfato de Adenosina/síntese química , Trifosfato de Adenosina/química , Animais , Humanos , Modelos Moleculares , Estrutura Molecular , Piperazinas/síntese química , Piperazinas/química , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Proteínas Proto-Oncogênicas c-akt/metabolismo , Pirimidinas/síntese química , Pirimidinas/química , Relação Estrutura-Atividade
11.
Nucleic Acids Res ; 44(6): e52, 2016 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-26657632

RESUMO

Stable isotope labeling is central to NMR studies of nucleic acids. Development of methods that incorporate labels at specific atomic positions within each nucleotide promises to expand the size range of RNAs that can be studied by NMR. Using recombinantly expressed enzymes and chemically synthesized ribose and nucleobase, we have developed an inexpensive, rapid chemo-enzymatic method to label ATP and GTP site specifically and in high yields of up to 90%. We incorporated these nucleotides into RNAs with sizes ranging from 27 to 59 nucleotides using in vitro transcription: A-Site (27 nt), the iron responsive elements (29 nt), a fluoride riboswitch from Bacillus anthracis(48 nt), and a frame-shifting element from a human corona virus (59 nt). Finally, we showcase the improvement in spectral quality arising from reduced crowding and narrowed linewidths, and accurate analysis of NMR relaxation dispersion (CPMG) and TROSY-based CEST experiments to measure µs-ms time scale motions, and an improved NOESY strategy for resonance assignment. Applications of this selective labeling technology promises to reduce difficulties associated with chemical shift overlap and rapid signal decay that have made it challenging to study the structure and dynamics of large RNAs beyond the 50 nt median size found in the PDB.


Assuntos
Trifosfato de Adenosina/síntese química , Guanosina Trifosfato/síntese química , Marcação por Isótopo/métodos , Nucleotídeos/síntese química , Bacillus anthracis/química , Bacillus anthracis/genética , Isótopos de Carbono , Coronavirus Humano 229E/química , Coronavirus Humano 229E/genética , Creatina Quinase/química , Creatina Quinase/genética , Espectroscopia de Ressonância Magnética , Pentosiltransferases/química , Pentosiltransferases/genética , Fosfotransferases (Aceptor do Grupo Álcool)/química , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Elementos de Resposta , Ribose/química , Ribose-Fosfato Pirofosfoquinase/química , Ribose-Fosfato Pirofosfoquinase/genética , Riboswitch , Transcrição Gênica
12.
ACS Chem Biol ; 10(11): 2544-52, 2015 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-26274552

RESUMO

Nucleotides containing adenosine play pivotal roles in every living cell. Adenosine triphosphate (ATP), for example, is the universal energy currency, and ATP-consuming processes also contribute to posttranslational protein modifications. Nevertheless, detecting the turnover of adenosine nucleotides in the complex setting of a cell remains challenging. Here, we demonstrate the use of fluorogenic analogs of ATP and adenosine tetraphosphate to study nucleotide hydrolysis in lysates of human cell lines and in intact human cells. We found that the adenosine triphosphate analog is completely stable in lysates of human cell lines, whereas the adenosine tetraphosphate analog is rapidly turned over. The observed activity in human cell lysates can be assigned to a single enzyme, namely, the human diadenosine tetraphosphate hydrolase NudT2. Since NudT2 has been shown to be a prognostic factor for breast cancer, the adenosine tetraphosphate analog might contribute to a better understanding of its involvement in cancerogenesis and allow the straightforward screening for inhibitors. Studying hydrolysis of the analogs in intact cells, we found that electroporation is a suitable method to deliver nucleotide analogs into the cytoplasm and show that high FRET efficiencies can be detected directly after internalization. Time-dependent experiments reveal that adenosine triphosphate and tetraphosphate analogs are both processed in the cellular environment. This study demonstrates that these nucleotide analogs indeed bear the potential to be powerful tools for the exploration of nucleotide turnover in the context of whole cells.


Assuntos
Trifosfato de Adenosina/análogos & derivados , Extratos Celulares/química , Nucleotídeos/metabolismo , Trifosfato de Adenosina/síntese química , Trifosfato de Adenosina/metabolismo , Western Blotting , Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes/química , Células HeLa , Humanos , Estrutura Molecular , Monoéster Fosfórico Hidrolases/metabolismo
13.
Org Biomol Chem ; 13(31): 8550-5, 2015 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-26173047

RESUMO

Protein AMPylation is a posttranslational modification (PTM) defined as the transfer of an adenosine monophosphate (AMP) from adenosine triphosphate (ATP) to a hydroxyl side-chain of a protein substrate. One recently reported AMPylator enzyme, Vibrio outer protein S (VopS), plays a role in pathogenesis by AMPylation of Rho GTPases, which disrupts crucial signaling pathways, leading to eventual cell death. Given the resurgent interest in this modification, there is a critical need for chemical tools that better facilitate the study of AMPylation and the enzymes responsible for this modification. Herein we report the synthesis of 2-ethynyl-adenosine-5'-triphosphate () and its utilization as a non-radioactive chemical reporter for protein AMPylation.


Assuntos
Monofosfato de Adenosina/metabolismo , Trifosfato de Adenosina/química , Trifosfato de Adenosina/síntese química , Corantes Fluorescentes/química , Corantes Fluorescentes/síntese química , Processamento de Proteína Pós-Traducional , Trifosfato de Adenosina/análogos & derivados , Biotina/química , Biotina/metabolismo , Técnicas de Química Sintética , Desenho de Fármacos , Proteína cdc42 de Ligação ao GTP/química , Proteína cdc42 de Ligação ao GTP/metabolismo
14.
Elife ; 42015 May 19.
Artigo em Inglês | MEDLINE | ID: mdl-25986605

RESUMO

Two ER membrane-resident transmembrane kinases, IRE1 and PERK, function as stress sensors in the unfolded protein response. IRE1 also has an endoribonuclease activity, which initiates a non-conventional mRNA splicing reaction, while PERK phosphorylates eIF2α. We engineered a potent small molecule, IPA, that binds to IRE1's ATP-binding pocket and predisposes the kinase domain to oligomerization, activating its RNase. IPA also inhibits PERK but, paradoxically, activates it at low concentrations, resulting in a bell-shaped activation profile. We reconstituted IPA-activation of PERK-mediated eIF2α phosphorylation from purified components. We estimate that under conditions of maximal activation less than 15% of PERK molecules in the reaction are occupied by IPA. We propose that IPA binding biases the PERK kinase towards its active conformation, which trans-activates apo-PERK molecules. The mechanism by which partial occupancy with an inhibitor can activate kinases may be wide-spread and carries major implications for design and therapeutic application of kinase inhibitors.


Assuntos
Trifosfato de Adenosina/farmacologia , Endorribonucleases/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Resposta a Proteínas não Dobradas/efeitos dos fármacos , eIF-2 Quinase/antagonistas & inibidores , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/síntese química , Animais , Bioensaio , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Estresse do Retículo Endoplasmático , Endorribonucleases/genética , Endorribonucleases/metabolismo , Ativação Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/enzimologia , Expressão Gênica , Genes Reporter , Células HEK293 , Humanos , Camundongos , Mimetismo Molecular , Inibidores de Proteínas Quinases/síntese química , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Transcrição de Fator Regulador X , Radioisótopos de Enxofre , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Resposta a Proteínas não Dobradas/genética , eIF-2 Quinase/genética , eIF-2 Quinase/metabolismo
15.
Curr Protoc Nucleic Acid Chem ; 60: 13.14.1-13.14.25, 2015 Mar 09.
Artigo em Inglês | MEDLINE | ID: mdl-25754889

RESUMO

This unit describes the synthesis of γ-phosphate-labeled and doubly labeled adenosine triphosphate (ATP) analogs and their characterization using the phosphodiesterase I from Crotalus adamanteus (snake venom phosphodiesterase; SVPD). In the key step of the synthesis, ATP or an ATP analog, bearing a linker containing a trifluoroacetamide group attached to the nucleoside, are modified with an azide-containing linker at the terminal phosphate using an alkylation reaction. Subsequently, different labels are introduced to the linkers by transformation of one functional group to an amine and coupling to an N-hydroxysuccinimide ester. Specifically, the Staudinger reaction of the azide is employed as a straightforward means to obtain an amine in the presence of various labels. Furthermore, the fluorescence characteristics of a fluorogenic, doubly labeled ATP analog are investigated following enzymatic cleavage by SVPD.


Assuntos
Trifosfato de Adenosina/síntese química , Nucleosídeos/química , Fosfatos/química , Coloração e Rotulagem , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/química , Azidas/química , Fluorescência , Nucleosídeos/síntese química , Diester Fosfórico Hidrolases/química , Succinimidas
16.
J Med Chem ; 57(14): 5919-34, 2014 Jul 24.
Artigo em Inglês | MEDLINE | ID: mdl-24972256

RESUMO

NTPDase2 catabolizes nucleoside triphosphates and consequently, through the interaction of nucleotides with P2 receptors, controls multiple biological responses. NTPDase2 inhibitors could modulate responses induced by nucleotides in thrombosis, inflammation, cancer, etc. Here we developed a set of ATP analogues as potential NTPDase inhibitors and identified a subtype-selective and potent NTPDase2 inhibitor, 2-hexylthio-ß,γ-methylene-ATP, 2. Analogue 2 was stable to hydrolysis by NTPDase1, -2, -3, and -8. It inhibited hNTPDase2 with Ki 20 µM, while only marginally (5-15%) inhibiting NTPDase1, -3, and -8. Homology models of hNTPDase1 and -2 were constructed. Docking and subsequent linear interaction energy (LIE) simulations provided a correlation with r2=0.94 between calculated and experimental inhibition data for the triphosphate analogues considered in this work. The origin of selectivity of 2 for NTPDase2 over NTPDase1 is the thiohexyl moiety of 2 which is favorably located within a hydrophobic pocket, whereas in NTPDase1 it is exposed to the solvent.


Assuntos
Adenosina Trifosfatases/antagonistas & inibidores , Trifosfato de Adenosina/análogos & derivados , Inibidores Enzimáticos/farmacologia , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/síntese química , Trifosfato de Adenosina/química , Trifosfato de Adenosina/farmacologia , Relação Dose-Resposta a Droga , Desenho de Fármacos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Modelos Moleculares , Estrutura Molecular , Relação Estrutura-Atividade
17.
J Med Chem ; 57(11): 4677-91, 2014 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-24846781

RESUMO

Aberrant nucleotide pyrophosphatase/phosphodiesterase-1 (NPP1) activity is associated with chondrocalcinosis, osteoarthritis, and type 2 diabetes. The potential of NPP1 inhibitors as therapeutic agents, and the scarceness of their structure-activity relationship, encouraged us to develop new NPP1 inhibitors. Specifically, we synthesized ATP-α-thio-ß,γ-CH2 (1), ATP-α-thio-ß,γ-CCl2 (2), ATP-α-CH2-γ-thio (3), and 8-SH-ATP (4) and established their resistance to hydrolysis by NPP1,3 and NTPDase1,2,3,8 (<5% hydrolysis) (NTPDase = ectonucleoside triphosphate diphosphohydrolase). Analogues 1-3 at 100 µM inhibited thymidine 5'-monophosphate p-nitrophenyl ester hydrolysis by NPP1 and NPP3 by >90% and 23-43%, respectively, and only slightly affected (0-40%) hydrolysis of ATP by NTPDase1,2,3,8. Analogue 3 is the most potent NPP1 inhibitor currently known, Ki = 20 nM and IC50 = 0.39 µM. Analogue 2a is a selective NPP1 inhibitor with Ki = 685 nM and IC50 = 0.57 µM. Analogues 1-3 were found mostly to be nonagonists of P2Y1/P2Y2/P2Y11 receptors. Docking analogues 1-3 into the NPP1 model suggested that activity correlates with the number of H-bonds with binding site residues. In conclusion, we propose analogues 2a and 3 as highly promising NPP1 inhibitors.


Assuntos
Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/síntese química , Organofosfatos/síntese química , Organofosfonatos/síntese química , Organotiofosfatos/síntese química , Pirofosfatases/antagonistas & inibidores , Trifosfato de Adenosina/farmacologia , Animais , Sítios de Ligação , Células COS , Domínio Catalítico , Linhagem Celular Tumoral , Chlorocebus aethiops , Humanos , Ligação de Hidrogênio , Hidrólise , Simulação de Acoplamento Molecular , Organofosfatos/farmacologia , Organofosfonatos/farmacologia , Organotiofosfatos/farmacologia , Diester Fosfórico Hidrolases , Agonistas do Receptor Purinérgico P2Y/síntese química , Agonistas do Receptor Purinérgico P2Y/farmacologia , Estereoisomerismo , Relação Estrutura-Atividade
18.
Se Pu ; 32(11): 1275-9, 2014 Nov.
Artigo em Chinês | MEDLINE | ID: mdl-25764665

RESUMO

A new method was developed for the determination of adenosine disodium triphos- phate (ATP-Na2 ) and its related substances in ATP-Na2 preparation by ion chromatography (IC). The sample was diluted with ultrapure water and filtrated by 0.22 µm polyether sulfone filter membrance, and then analyzed by IC directly without any more pretreatment. The analysis was performed on a Dionex IonPac AS11-HC column (250 mm x 4 mm) and a guard column Ion-Pac AG11-HC (50 mm x 4 mm). A KOH eluent generator cartridge was used for gradient elution at the flow rate of 1.0 mL/min. The detection was performed by a Dionex suppressed (Dionex AERS 500 4-mm) conductivity detector. The injection volume was 10 µL. The assay was quantitatively completed by external standard method and the related substances were calculated with correction factors. The linear ranges of the method for ATP-Na2, adenosine disodium diphosphate (ADP-Na2) and adenosine disodium monophosphate (AMP-Na2) were 0.000 146-1.83 g/L (r = 0.9997), 0.000484-1.51 g/L (r = 0.9996) and 0.000426-0.804 g/L (r = 0.9999), respectively. The average recoveries of ATP-Na2 were 96.50%, 96.57% and 96.77% at three spiked levels. The limits of quantitation (S/N = 10) of ATP-Na2, ADP-Na2 and AMP-Na2 were 1.5 ng, 4.8 ng, 4.3 ng, and the limits of detection (S/N = 3) were 0.58 ng, 1.21 ng, 1.28 ng, respectively. The results demonstrated that the system has the advantages of high sensitivity, facile automation and simple sample pretreatment. The method is suitable for the quality control of adenosine disodium triphosphate preparations.


Assuntos
Trifosfato de Adenosina/química , Trifosfato de Adenosina/síntese química , Cromatografia por Troca Iônica , Análise de Componente Principal , Água
19.
J Mol Evol ; 76(5): 332-42, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23689512

RESUMO

A model for abiotic photophosphorylation of adenosine diphosphate by orthophosphate with the formation of adenosine triphosphate was studied. The model was based on the photochemical activity of the abiogenic conjugates of pigments with the polymeric material formed after thermolysis of amino acid mixtures. The pigments formed showed different fluorescence parameters depending on the composition of the mixture of amino acid precursors. Thermolysis of the mixture of glutamic acid, glycine, and lysine (8:3:1) resulted in a predominant formation of a pigment fraction which had the fluorescence maximum at 525 nm and the excitation band maxima at 260, 375, and 450 nm and was identified as flavin. When glycine in the initial mixture was replaced with alanine, a product formed whose fluorescence parameters were typical to pteridines (excitation maximum at 350 nm, emission maximum at 440 nm). When irradiated with the quasi-monochromatic light (over the range 325-525 nm), microspheres in which flavin pigments were prevailing showed a maximum photophosphorylating activity at 375 and 450 nm, and pteridine-containing chromoproteinoid microspheres were most active at 350 nm. The positions and the relative height of maxima in the action spectra correlate with those in the excitation spectra of the pigments, which point to the involvement of abiogenic flavins and pteridines in photophosphorylation.


Assuntos
Difosfato de Adenosina/química , Trifosfato de Adenosina/síntese química , Aminoácidos/química , Flavinas/síntese química , Fosfatos/química , Pigmentos Biológicos/síntese química , Pteridinas/síntese química , Temperatura Alta , Luz , Fotofosforilação , Espectrometria de Fluorescência , Termodinâmica
20.
Br J Pharmacol ; 169(1): 179-96, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23425137

RESUMO

BACKGROUND AND PURPOSE: Ectonucleotidases control extracellular nucleotide levels and consequently, their (patho)physiological responses. Among these enzymes, nucleoside triphosphate diphosphohydrolase-1 (NTPDase1), -2, -3 and -8 are the major ectonucleotidases responsible for nucleotide hydrolysis at the cell surface under physiological conditions, and NTPDase1 is predominantly located at the surface of vascular endothelial cells and leukocytes. Efficacious inhibitors of NTPDase1 are required to modulate responses induced by nucleotides in a number of pathological situations such as thrombosis, inflammation and cancer. EXPERIMENTAL APPROACH: Here, we present the synthesis and enzymatic characterization of five 8-BuS-adenine nucleotide derivatives as potent and selective inhibitors of NTPDase1. KEY RESULTS: The compounds 8-BuS-AMP, 8-BuS-ADP and 8-BuS-ATP inhibit recombinant human and mouse NTPDase1 by mixed type inhibition, predominantly competitive with Ki values <1 µM. In contrast to 8-BuS-ATP which could be hydrolyzed by other NTPDases, the other BuS derivatives were resistant to hydrolysis by either NTPDase1, -2, -3 or -8. 8-BuS-AMP and 8-BuS-ADP were the most potent and selective inhibitors of NTPDase1 expressed in human umbilical vein endothelial cells as well as in situ in human and mouse tissues. As expected, as a result of their inhibition of recombinant human NTPDase1, 8-BuS-AMP and 8-BuS-ADP impaired the ability of this enzyme to block platelet aggregation. Importantly, neither of these two inhibitors triggered platelet aggregation nor prevented ADP-induced platelet aggregation, in support of their inactivity towards P2Y1 and P2Y12 receptors. CONCLUSIONS AND IMPLICATIONS: The 8-BuS-AMP and 8-BuS-ADP have therefore potential to serve as drugs for the treatment of pathologies regulated by NTPDase1.


Assuntos
Difosfato de Adenosina/farmacologia , Monofosfato de Adenosina/farmacologia , Trifosfato de Adenosina/farmacologia , Apirase/antagonistas & inibidores , Difosfato de Adenosina/análogos & derivados , Difosfato de Adenosina/síntese química , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/síntese química , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/síntese química , Animais , Antígenos CD , Células COS , Chlorocebus aethiops , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Camundongos , Agregação Plaquetária/efeitos dos fármacos , Ratos , Especificidade da Espécie
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