Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 76
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Curr Protoc ; 1(11): e287, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34826354

RESUMO

This article presents a new method for the rapid, stereoselective and regioselective synthesis of nucleoside 2',3'-O,O-phosphorothioate and 2',3'-O,O-phosphoroselenoate molecules. The method avoids the use of protection groups, chiral reagents, and chiral metal catalysts, as well as complicated chiral separations. This synthetic method has been applied successfully to all of the natural nucleosides. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Preparation of 2-chloro-1,3,2-dithiophospholane (6) Basic Protocol 2: Preparation of 2-cyanoethoxy-thio-dithiophospholane (8) Basic Protocol 3: Preparation of 2-cyanoethoxy-seleno-dithiophospholane (9) Basic Protocol 4: Preparation of uridine-2',3'-O,O-phosphorothioate (Sp, exo; 1A) Basic Protocol 5: Preparation of uridine-2',3'-O,O-phosphoroselenoate (Sp, exo; 11) Basic Protocol 6: Preparation of adenosine-2',3'-O,O-phosphorothioate (Sp, exo; 12) Basic Protocol 7: Preparation of thymidine-2',3'-O,O-phosphorothioate (Sp, exo; 13) Basic Protocol 8: Preparation of cytosine-2',3'-O,O-phosphorothioate (Sp, exo; 14) Basic Protocol 9: Preparation of guanosine-2',3'-O,O-phosphorothioate (Sp, exo; 15).


Assuntos
Nucleosídeos , Catálise , Timidina , Uridina
2.
ACS Omega ; 5(48): 31314-31322, 2020 Dec 08.
Artigo em Inglês | MEDLINE | ID: mdl-33324842

RESUMO

Uridine (U) mimetics are sought after as tools for biochemical and pharmacological studies. Previously, we have identified recognition patterns of U by proteins. Here, we targeted the characterization of uridine mimetics-cyanuryl-ribose (CR), barbituryl-ribose (BR), and 6-azauridine (AU)-with a view to identify analogs with potentially more binding interactions than U with target biomolecules. We found that CR, BR, and AU retain selective U's natural H-bonds with adenosine vs guanosine. CR/AU and BR were 100- and 10,000-fold more acidic, respectively, than U. Under physiological pH, 54, 51, and 77% of CR, AU, and BR molecules, respectively, are ionized vs 13% for U. The electron-rich nature of CR and BR vs U was reflected by their 13C NMR chemical shifts and ε values. CR/AU and BR prefer N conformation (up to 73%) vs U (56%). Unlike U that prefers gg conformation around exocyclic methylol (48%), CR/AU and BR prefer both gt and gg rotamers. In conclusion, replacement of uridine's C6 by N or carbonyl, or C5-C6 by an amide, results in significant changes in U's ionization, electron density, conformation, base-stacking, etc., leading to potentially tighter binding than U with a target protein or nucleic acid and potential use for various biochemical and pharmacological applications.

3.
Chem Commun (Camb) ; 56(78): 11633-11636, 2020 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-32870217

RESUMO

A new facile, rapid, stereo- and regio-selective one-pot synthesis of nucleoside-2',3'-O,O-phosphorothioate and selenoate analogs has been developed. This method avoids the need for protection strategies and chiral reagents, chiral metal catalysts, or chiral separations. This synthetic method has been applied to all natural nucleosides (U/A/G/C/T). Furthermore, we have deciphered the origin of the stereo- and regio-selectivity of the reaction.

4.
Bioconjug Chem ; 31(4): 1188-1198, 2020 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-32208683

RESUMO

Currently, there is demand for fluorescent oligonucleotide probes for diagnostic purposes. To address this necessity, we developed nucleosides containing a flexible spacer with an intercalating moiety at its end (NIC molecules). The intercalator is based on 4-hydroxybenzylidene imidazolinone (HBI), found in the Green Fluorescent Protein. We synthesized 20-mer oligonucleotides, ON1-ON4, incorporating the DMTr phosphorodiamidite monomer of dUHBI, 2, and the corresponding dUDFHBI, 5b, monomer. ON1-ON4 target the HER-2 mRNA breast cancer marker for the diagnostics of breast cancer subtype. Hybridization of ON1/ON2 and ON3/ON4 with complementary 2'-OMe-RNA resulted in emission at 462 and 481 nm, respectively, and up to 46-fold increase in fluorescence intensity. CD and 19F-NMR data indicated that HBI and DFHBI fluorophores bind as intercalators and stabilize the duplexes (up to ΔTm 6 °C). Furthermore, addition of ON1-ON4 to total RNA extracted from cancer cells that overexpress HER-2 mRNA, resulted in a significant fluorescence enhancement of ON3 and ON4. The latter sensitively detected low concentrations of the target mRNA (at total RNA 30 ng/µL). These probes were photostable for 200 min. Using a dilution curve, we quantified the number of HER-2 transcripts in a cell. In conclusion, ON3 and ON4 are promising diagnostic probes for an easy, instantaneous, specific, and sensitive detection of levels of oncogenes. Importantly, the NIC concept, demonstrated here for diagnostics of breast cancer, is universal and may be applied not only in a clinical setting but also for the detection of any RNA.


Assuntos
Biomarcadores Tumorais/genética , Neoplasias da Mama/genética , Corantes Fluorescentes/química , Limite de Detecção , Receptor ErbB-2/genética , Linhagem Celular Tumoral , Humanos , Hibridização de Ácido Nucleico , RNA Mensageiro/química , RNA Mensageiro/genética
5.
Curr Protoc Nucleic Acid Chem ; 80(1): e104, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32032480

RESUMO

Nucleoside intercalator conjugates (NICs) describe an innovative methodology developed in our research group for preparation of fluorescence turn-on DNA hybridization probes targeting specific mRNA sequences (e.g., breast cancer markers). In this methodology, we conjugate a non-fluorescent intercalator to the base of a nucleic acid (e.g., uracil) via a flexible spacer. This modified monomer can be incorporated into oligonucleotides by solid-phase synthesis and a large fluorescence enhancement is observed when the modified oligonucleotide is hybridized with its complementary strand due to intercalation of the fluorophore between the two strands. 5-(6-p-Methoxybenzylidene imidazolinone-1-hexene)-2'-deoxyuridine (dUMBI ) is a synthetic monomer to which 4-methoxybenzylidene imidazolinone (MBI), the fluorescent chromophore of green fluorescent protein (GFP), has been conjugated via a flexible spacer. The detection of human epidermal growth factor receptor 2 (HER2) mRNA by this probe has already been established by our group. The fluorescent intensity of the single-strand DNA can be considered as negligible due to the free rotation of the fluorophore. Upon hybridization, however, the flexible spacer allows for the intercalation of the fluorophore between the hybridized strands, giving rise to enhanced fluorescence and indicating the presence of target mRNA. 3,5-Difluoro-4-methoxybenzylidene (DFMBI) has enhanced photophysical properties compared to MBI fluorophore. This protocol describes a simple, reliable, efficient, and general method for the synthesis of improved derivative dUDFMBI as a monomer of fluorescent turn-on DNA hybridization probe with application for detection of HER2 mRNA. © 2020 by John Wiley & Sons, Inc. Basic Protocol: Synthesis of 5-[(6)-3,5-difluoro-4-methoxybenzylidene imidazolinone-1-hexene]-2'-deoxyuridine.


Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/metabolismo , Desoxiuridina/síntese química , Imidazolinas/química , Sondas de Oligonucleotídeos/química , Receptor ErbB-2/metabolismo , Desoxiuridina/química , Feminino , Humanos
6.
Org Biomol Chem ; 17(46): 9913-9923, 2019 11 27.
Artigo em Inglês | MEDLINE | ID: mdl-31720670

RESUMO

Nucleotide pyrophosphatase/phosphodiesterase-1 (NPP1) inhibitors have been suggested as a potential treatment for calcium pyrophosphate dihydrate (CPPD) deposition disease. Here, we targeted the development of improved NPP1 inhibitors based on acyclic mimics of Pα,α-phosphorodithioate-substituted adenine nucleotides, 7-10. The latter were obtained in a facile two-step synthesis from adenine-(methoxy)ethanol. Among analogs 7-10, adenine-(methoxy)ethoxy-Pα,α-dithio-triphosphate, 8, was the most potent NPP1 inhibitor both with purified enzyme (IC50 0.645 µM) and in osteoarthritic human chondrocytes (IC50 0.033 µM). Furthermore, it efficaciously (10-fold vs. control) inhibited ATP-induced CPPD in human articular chondrocytes. Importantly, 8 was a highly selective NPP1 inhibitor which showed only minor inhibition of NPP3, CD39 and CD73, and did not inhibit TNAP (tissue nonspecific alkaline phosphatase) activity in human chondrocytes. Furthermore, 8 did not activate P2Y1,2,6 receptors. Analog 8 was not toxic to cultured chondrocytes at 100 µM. Therefore, 8 may be suitable for further development as a drug candidate for the treatment of CPPD arthritis and other NPP1-related diseases.


Assuntos
Adenina/farmacologia , Pirofosfato de Cálcio/antagonistas & inibidores , Condrócitos/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Osteoartrite do Joelho/tratamento farmacológico , Polifosfatos/farmacologia , Pirofosfatases/antagonistas & inibidores , Compostos de Sulfidrila/farmacologia , Adenina/síntese química , Adenina/química , Pirofosfato de Cálcio/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Condrócitos/metabolismo , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Estrutura Molecular , Osteoartrite do Joelho/metabolismo , Osteoartrite do Joelho/patologia , Diester Fosfórico Hidrolases/metabolismo , Polifosfatos/química , Pirofosfatases/metabolismo , Relação Estrutura-Atividade , Compostos de Sulfidrila/química
7.
Eur J Med Chem ; 184: 111754, 2019 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-31610377

RESUMO

Overexpression of ecto-nucleotide pyrophosphatase-1 (NPP1) is associated with diseases such as calcium pyrophosphate dihydrate deposition disease, calcific aortic valve disease, and type 2 diabetes. In this context, NPP1 inhibitors are potential drug candidates for the treatment of these diseases. The present study focuses on the analysis of the structure-activity relationship of NPP1 inhibitors based on acyclic uracil-nucleotides. For this purpose, we synthesized acyclic uridine-monophosphate analogs, 10-11, uridine-diphosphate analogs, 12-14, and uridine-Pα,α-dithio-triphosphate analogs, 15-17. We evaluated their inhibitory activity and selectivity towards NPP1, -3, NTPDase1, -2, -3, and -8, and P2Y2,4,6 receptors. Analogs 16 and 17 were the most selective and potent NPP1 inhibitors (Ki 0.94 and 0.73 µM, respectively) among the tested molecules. Analogs 10-17 had only minute effect on uracil-nucleotide responding P2Y2,4,6 receptors. Analog 17 (100 µM) displayed 96% inhibition of NPPase activity in osteoarthritic human chondrocytes. Analogs 14-17 displayed weak inhibitory effect on alkaline phosphatase activity at equimolar concentrations in human chondrocytes. All tested analogs showed no toxicity at human chondrocytes. We concluded that ribose-ring to chain transformation, as well as the type of the nucleobase, are parameters of minor significance to NPP1 inhibition, whereas the major parameter is Pα-dithio-substitution. In addition, the length of the phosphate chain also significantly affects inhibition. Overall, the experimental results were well reproduced by molecular docking. A correlation was observed between the activities of the compounds and the number of H-bonds and salt bridges formed between the inhibitors and NPP1 binding site residues. Uracil-N1-(methoxy)ethyl-ß-Pα,α-dithio, Pß,γ-methylene tri-phosphate, 17, was identified as the most potent, selective, and non-toxic NPP1 inhibitor among the tested analogs, and may be used as a lead structure for further drug development.


Assuntos
Organofosfatos/farmacologia , Inibidores de Fosfodiesterase/farmacologia , Pirofosfatases/antagonistas & inibidores , Uracila/farmacologia , Condrócitos/efeitos dos fármacos , Condrócitos/metabolismo , Relação Dose-Resposta a Droga , Humanos , Estrutura Molecular , Organofosfatos/síntese química , Organofosfatos/química , Inibidores de Fosfodiesterase/síntese química , Inibidores de Fosfodiesterase/química , Diester Fosfórico Hidrolases/metabolismo , Pirofosfatases/metabolismo , Relação Estrutura-Atividade , Uracila/síntese química , Uracila/química
10.
Inorg Chem ; 58(14): 8995-9003, 2019 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-31247811

RESUMO

Recently, we reported on a series of aminomethylene-phosphonate (AMP) analogues, bearing one or two heterocyclic groups on the aminomethylene moiety, as promising Zn(II) chelators. Given the strong Zn(II) binding properties of these compounds, they may find useful applications in metal chelation therapy. With a goal of inhibiting the devastating oxidative damage caused by prion protein in prion diseases, we explored the most promising ligand, {bis[(1H-imidazol-4-yl)methyl]amino}methylphosphonic acid, AMP-(Im)2, 4, as an inhibitor of the oxidative reactivity associated with the Cu(II) complex of prion peptide fragment 84-114. Specifically, we first characterized the Cu(II) complex with AMP-(Im)2 by ultraviolet-visible spectroscopy and electrochemical measurements that indicated the high chemical and electrochemical stability of the complex. Potentiometric pH titration provided evidence of the formation of a stable 1:1 [Cu(II)-AMP-(Im)2]+ complex (ML), with successive binding of a second AMP-(Im)2 molecule yielding ML2 complex [Cu(II)-(AMP-(Im)2)2]+ (log K' = 15.55), and log ß' = 19.84 for ML2 complex. The CuN3O1 ML complex was demonstrated by X-ray crystallography, indicating the thermodynamically stable square pyramidal complex. Chelation of Cu(II) by 4 significantly reduced the oxidation potential of the former. CuCl2 and the 1:2 Cu:AMP-(Im)2 complex showed one-electron redox of Cu(II)/Cu(I) at 0.13 and -0.35 V, respectively. Indeed, 4 was found to be a potent antioxidant that at a 1:1:1 AMP-(Im)2:Cu(II)-PrP84-114 molar ratio almost totally inhibited the oxidation reaction of 4-methylcatechol. Circular dichroism data suggest that this antioxidant activity is due to formation of a ternary, redox inactive Cu(II)-Prp84-114-[AMP-(Im)2] complex. Future studies in prion disease animal models are warranted to assess the potential of 4 to inhibit the devastating oxidative damage caused by PrP.


Assuntos
Cobre/química , Isoxazóis/química , Príons/química , Tetrazóis/química , Modelos Moleculares , Oxirredução , Conformação Proteica
11.
Purinergic Signal ; 15(2): 247-263, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31025169

RESUMO

Overproduction of extracellular diphosphate due to hydrolysis of ATP by NPP1 leads to pathological calcium diphosphate (pyrophosphate) dihydrate deposition (CPPD) in cartilage, resulting in a degenerative joint disease that today lacks a cure. Here, we targeted the identification of novel NPP1 inhibitors as potential therapeutic agents for CPPD deposition disease. Specifically, we synthesized novel analogs of AMP (NPP1 reaction product) and ADP (NPP1 inhibitor). These derivatives incorporate several chemical modifications of the natural nucleotides including (1) a methylene group replacing the Pα,ß-bridging oxygen atom to provide metabolic resistance, (2) sulfonate group(s) replacing phosphonate(s) to improve binding to NPP1's catalytic zinc ions, (3) an acyclic nucleotide analog to allow flexible binding in the NPP1 catalytic site, and (4) a benzimidazole base replacing adenine. Among the investigated compounds, adenine-N9-(methoxy)ethyl-ß-bisphosphonate, 10, was identified as an NPP1 inhibitor (Ki 16.3 µM vs. the artificial substrate p-nitrophenyl thymidine-5'-monophosphate (p-Nph-5'-TMP), and 9.60 µM vs. the natural substrate, ATP). Compound 10 was selective for NPP1 vs. human NPP3, human CD39, and tissue non-specific alkaline phosphatase (TNAP), but also inhibited human CD73 (Ki 12.6 µM). Thus, 10 is a dual NPP1/CD73 inhibitor, which could not only be of interest for treating CPPD deposition disease and calcific aortic valve disease but may also be considered for the immunotherapy of cancer. Compound 10 proved to be a promising inhibitor, which almost completely reduces NPPase activity in human osteoarthritic chondrocytes at a concentration of 100 µM.


Assuntos
Difosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/análogos & derivados , Inibidores Enzimáticos/farmacologia , Pirofosfatases/antagonistas & inibidores , Condrocalcinose , Condrócitos/efeitos dos fármacos , Humanos , Osteoartrite , Diester Fosfórico Hidrolases
12.
Eur J Med Chem ; 173: 99-106, 2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-30991278

RESUMO

Diagnosis and treatment of breast cancer can be greatly enhanced and personalized based on the quantitative detection of mRNA markers. Here, we targeted the development of a fluorescent oligonucleotide probe to detect specifically the HER-2 mRNA breast cancer marker. We have selected the chromophore of the Green Fluorescent Protein (GFP), 4-hydroxybenzylidene imidazolinone (HBI), as a fluorophore covalently bound to an oligonucleotide probe and potentially capable of intercalating within a probe-mRNA duplex. We first synthesized the two-ring scaffold of the HBI chromophore 5 and coupled it to 2'-deoxyuridine at C5-position via a 7-atom-spacer, to give 4. Indeed, in the highly viscous glycerol used to mimic the reduced conformational flexibility of the intercalated HBI, chromophore 4 displayed a quantum yield of 0.29 and brightness of 20600 M-1cm-1, while no fluorescent signal was observed in methanol. Next, we synthesized a 20-mer oligonucleotide probe incorporating 4 at position 6 (5'-CCCGTUTCAACAGGAGTTTC-3'), ONHBI, targeting nucleotides 1233-1253 of HER-2 mRNA. A 16-fold enhancement of ONHBI emission intensity upon hybridization with the complementary RNA vs that of the oligonucleotide probe alone indicated the presence of target oligonucleotide and proved the intercalation of the chromophore (quantum yield 0.52; brightness 23500 M-1cm-1). Even more, an 11-fold enhancement of ONHBI emission (quantum yield 0.50; brightness 23200 M-1cm-1) was observed when the probe was mixed with total RNA extract from a human cell line that has high levels of HER2 mRNA expression. Thus, we propose ONHBI as a promising probe potentially useful for the sensitive and specific detection of HER2 mRNA breast cancer marker.


Assuntos
Biomarcadores Tumorais/análise , Neoplasias da Mama/diagnóstico por imagem , Proteínas de Fluorescência Verde/química , Sondas de Oligonucleotídeos/química , RNA Mensageiro/análise , Receptor ErbB-2/análise , Linhagem Celular Tumoral , Feminino , Humanos , Estrutura Molecular , Sondas de Oligonucleotídeos/síntese química , Espectrometria de Fluorescência
13.
Purinergic Signal ; 14(3): 271-284, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30019187

RESUMO

Extracellular nucleotides can regulate the production/drainage of the aqueous humor via activation of P2 receptors, thus affecting the intraocular pressure (IOP). We evaluated 5-OMe-UDP(α-B), 1A, a potent P2Y6-receptor agonist, for reducing IOP and treating glaucoma. Cell viability in the presence of 1A was measured using [3-(4, 5-dimethyl-thiazol-2-yl) 2, 5-diphenyl-tetrazolium bromide] (MTT) assay in rabbit NPE ciliary non-pigmented and corneal epithelial cells, human retinoblastoma, and liver Huh7 cells. The effect of 1A on IOP was determined in acute glaucomatous rabbit hyaluronate model and phenol-induced chronic glaucomatous rabbit model. The origin of activity of 1A was investigated by generation of a homology model of hP2Y6-R and docking studies. 1A did not exert cytotoxic effects up to 100 mM vs. trusopt and timolol in MTT assay in ocular and liver cells. In normotensive rabbits, 100 µM 1A vs. xalatan, trusopt, and pilocarpine reduced IOP by 45 vs. 20-30%, respectively. In the phenol animal model, 1A (100 µM) showed reduction of IOP by 40 and 20%, following early and late administration, respectively. Docking results suggest that the high activity and selectivity of 1A is due to intramolecular interaction between Pα-BH3 and C5-OMe which positions 1A in a most favorable site inside the receptor. P2Y6-receptor agonist 1A effectively and safely reduces IOP in normotense, acute, and chronic glaucomatous rabbits, and hence may be suggested as a novel approach for the treatment of glaucoma.


Assuntos
Glaucoma , Pressão Intraocular/efeitos dos fármacos , Agonistas do Receptor Purinérgico P2Y/farmacologia , Receptores Purinérgicos P2/efeitos dos fármacos , Animais , Humanos , Coelhos , Difosfato de Uridina/química , Difosfato de Uridina/farmacologia
14.
J Med Chem ; 61(9): 3939-3951, 2018 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-29681152

RESUMO

Ectonucleotide pyrophosphatase/phosphodiesterase-1 (NPP1) hydrolyzes phosphodiester bonds of nucleotides such as ATP, resulting mainly in the formation of AMP and pyrophosphate. NPP1 activity plays a deleterious function in calcified aortic valve disease and calcium pyrophosphate deposition disease. Thus, inhibitors of NPP1 represent a medical need. We developed novel NPP1 inhibitors based on uridine 5'-Pα,α-dithiophosphate analogues, 9-12. All these analogues potently inhibited hNPP1 (80-100% inhibition) at 100 µM, with no, or minimal, inhibition of NPP3 and other ectonucleotidases (NTPDase1,2,3,8). These compounds showed nearly no activity at uracil-nucleotide sensitive P2Y2,4,6-receptors and thus represent highly selective NPP1 inhibitors. The most promising inhibitor was diuridine 5'-Pα,α,5″-Pα,α-tetrathiotetraphosphate, 12, exhibiting Ki of 27 nM. Analogues 9-12 proved to be highly stable to air oxidation and to acidic and basic pH. Docking simulations suggested that the enhanced NPP1 inhibitory activity and selectivity of analogue 12 could be attributed to the simultaneous occupancy of two sites (the AMP site and an alternative site) of NPP1 by this compound.


Assuntos
Desenho de Fármacos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Pirofosfatases/antagonistas & inibidores , Nucleotídeos de Uracila/química , Nucleotídeos de Uracila/farmacologia , Estabilidade de Medicamentos , Inibidores Enzimáticos/metabolismo , Humanos , Hidrólise , Concentração Inibidora 50 , Simulação de Acoplamento Molecular , Diester Fosfórico Hidrolases/química , Diester Fosfórico Hidrolases/metabolismo , Conformação Proteica , Pirofosfatases/química , Pirofosfatases/metabolismo , Relação Estrutura-Atividade , Especificidade por Substrato , Nucleotídeos de Uracila/metabolismo
15.
Rheumatology (Oxford) ; 57(8): 1472-1480, 2018 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-29688536

RESUMO

Objectives: Calcium pyrophosphate deposition (CPPD) is associated with osteoarthritis and is the cause of a common inflammatory articular disease. Ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (eNPP1) is the major ecto-pyrophosphatase in chondrocytes and cartilage-derived matrix vesicles (MVs). Thus, eNPP1 is a principle contributor to extracellular pyrophosphate levels and a potential target for interventions aimed at preventing CPPD. Recently, we synthesized and described a novel eNPP1-specific inhibitor, SK4A, and we set out to evaluate whether this inhibitor attenuates nucleotide pyrophosphatase activity in human OA cartilage. Methods: Cartilage tissue, chondrocytes and cartilage-derived MVs were obtained from donors with OA undergoing arthroplasty. The effect of SK4A on cell viability was assayed by the XTT method. eNPP1 expression was evaluated by western blot. Nucleotide pyrophosphatase activity was measured by a colorimetric assay and by HPLC analysis of adenosine triphosphate (ATP) levels. ATP-induced calcium deposition in cultured chondrocytes was visualized and quantified with Alizarin red S staining. Results: OA chondrocytes expressed eNPP1 in early passages, but this expression was subsequently lost upon further passaging. Similarly, significant nucleotide pyrophosphatase activity was only detected in early-passage chondrocytes. The eNPP1 inhibitor, SK4A, was not toxic to chondrocytes and stable in culture medium and human plasma. SK4A effectively inhibited nucleotide pyrophosphatase activity in whole cartilage tissue, in chondrocytes and in cartilage-derived MVs and reduced ATP-induced CPPD. Conclusion: Nucleotide analogues such as SK4A may be developed as potent and specific inhibitors of eNPP1 for the purpose of lowering extracellular pyrophosphate levels in human cartilage with the aim of preventing and treating CPPD disease.


Assuntos
Calcinose/tratamento farmacológico , Pirofosfato de Cálcio/metabolismo , Condrocalcinose/tratamento farmacológico , Condrócitos/patologia , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/farmacologia , Pirofosfatases/antagonistas & inibidores , Calcinose/metabolismo , Calcinose/patologia , Células Cultivadas , Condrocalcinose/metabolismo , Condrocalcinose/patologia , Condrócitos/efeitos dos fármacos , Condrócitos/metabolismo , Colorimetria , Humanos , Immunoblotting , Diester Fosfórico Hidrolases/biossíntese , Pirofosfatases/biossíntese
16.
J Alzheimers Dis ; 58(2): 389-400, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28453480

RESUMO

BACKGROUND: Metal-ion-chelation was suggested to prevent zinc and copper ions-induced amyloid-ß (Aß) aggregation and oxidative stress, both implicated in the pathophysiology of Alzheimer's disease (AD). In a quest for biocompatible metal-ion chelators potentially useful for AD therapy, we previously tested a series of nucleoside 5'-phosphorothioate derivatives as agents for decomposition of Cu(I)/Cu(II)/Zn(II)-Aß-aggregates, and as inhibitors of OH radicals formation in Cu(I) or Fe(II) /H2O2 solution. Specifically, in our recent study we have identified 2-SMe-ADP(α-S), designated as SAS, as a most promising neuroprotectant. OBJECTIVE: To further explore SAS ability to protect the brain from Aß toxicity both in vitro and in vivo. METHODS: We evaluated SAS ability to decompose or inhibit the formation of Aß42-M(II) aggregates, and rescue primary neurons and astrocytes from Aß42 toxicity. Furthermore, we aimed at exploring the therapeutic effect of SAS on behavioral and cognitive deficits in the 5XFAD mouse model of AD. RESULTS: We found that SAS can rescue primary culture of neurons and astrocytes from Aß42 toxicity and to inhibit the formation and dissolve Aß42-Zn(II)/Cu(II) aggregates. Furthermore, we show that SAS treatment can prevent behavioral disinhibition and ameliorate spatial working memory deficits in 5XFAD mice. Notably, the mice were treated at the age of 2 months, before the onset of AD symptoms, for a duration of 2 months, while the effect was demonstrated at the age of 6 months. CONCLUSION: Our results indicate that SAS has the potential to delay progression of core pathological characteristics of AD in the 5XFAD mouse model.


Assuntos
Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/patologia , Antipsicóticos/uso terapêutico , Materiais Biocompatíveis/uso terapêutico , Fenotiazinas/uso terapêutico , Adenosina/análogos & derivados , Adenosina/farmacologia , Doença de Alzheimer/genética , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Animais Recém-Nascidos , Antipsicóticos/química , Materiais Biocompatíveis/química , Células Cultivadas , Córtex Cerebral/citologia , Cobre/uso terapêutico , Modelos Animais de Doenças , L-Lactato Desidrogenase/metabolismo , Aprendizagem em Labirinto/fisiologia , Transtornos da Memória/etiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Agregação Plaquetária/efeitos dos fármacos , Presenilina-1/genética , Presenilina-1/metabolismo , Zinco/uso terapêutico
17.
Org Biomol Chem ; 14(20): 4640-53, 2016 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-27109038

RESUMO

Currently, there is an urgent need for biocompatible metal-ion chelators capable of antioxidant activity and disassembly of amyloid beta (Aß)-aggregates as potential therapeutics for Alzheimer's disease (AD). We recently demonstrated the promising antioxidant activity of adenine/guanine 2',3' or 3',5'-bis(thio)phosphate analogues, 2'-dA/G3'5'PO/S and A2'3'PO/S, and their affinity to Zn(ii)-ions. These findings encouraged us to evaluate them as agents for the dissolution of Aß42-Zn(ii)/Cu(ii) aggregates. Specifically, we explored their ability to bind Cu(ii)/Zn(ii)-ions, the geometry and stoichiometry of these complexes, Cu(ii)/Zn(ii)-binding-sites and binding mode, and the ability of these analogues to dissolve Aß42-Zn(ii)/Cu(ii) aggregates, as well as their effect on the secondary structure of those aggregates. Finally, we identified the most promising agents for dissolution of Aß42-Zn(ii)/Cu(ii) aggregates. Specifically, we observed the formation of a 1 : 1 complex between 2'-dG3'5'PO and Cu(ii), involving O4 ligands. Zn(ii) was coordinated by both thiophosphate groups of 2'-dA3'5'PS and A2'3'PS involving O2S2 ligands in a 1 : 1 stoichiometry. A2'3'PS dissolves Aß42-Zn(ii) and Aß42-Cu(ii) aggregates as effectively as, and 2.5-fold more effectively than EDTA, respectively. Furthermore, 2'-dG3'5'PS and A2'3'PS reverted the Aß42-M(ii) structure, back to that of the free Aß42. Finally, cryo-TEM and TEM images confirmed the disassembly of Aß42 and Aß42-M(ii) aggregates by A2'3'PS. Hence, 2'-dG3'5'PS and A2'3'PS may serve as promising scaffolds for new AD therapeutics, acting as both effective antioxidants and agents for solubilization of Aß42-Cu(ii)/Zn(ii) aggregates.


Assuntos
Peptídeos beta-Amiloides/química , Cobre/química , Nucleosídeos/química , Fragmentos de Peptídeos/química , Fosfatos/química , Fosfatos/farmacologia , Agregados Proteicos/efeitos dos fármacos , Zinco/química , Antioxidantes/química , Antioxidantes/farmacologia , Quelantes/química , Quelantes/farmacologia
18.
Dalton Trans ; 44(48): 21073-80, 2015 Dec 28.
Artigo em Inglês | MEDLINE | ID: mdl-26592926

RESUMO

Hybrids of methylenediphosphonotetrathioate and crown-ether (MDPT-CE) were synthesized forming 7-,8-,9-,10- and 13-membered rings. Both 7- and 13-membered ring-containing compounds were found to be highly stable to air-oxidation for at least four weeks. These hybrids bind Zn(II) by both MDPT and CE moieties, forming a 2 : 1 L : Zn(II) complex. Interestingly, the 13-membered ring MDPT-CE showing a high affinity to Zn(II) (Ka 3 ± 0.5 × 10(6) mol(-2) L(2)) does not bind Li(I) or Na(I). The 13-Membered MDPT-CE hybrid is a promising water-soluble, air-stable, high-affinity Zn(II)-chelator, exhibiting selectivity to Zn(II) vs. Mg(II), Na(I), and Li(I).

19.
J Med Chem ; 58(21): 8427-43, 2015 Nov 12.
Artigo em Inglês | MEDLINE | ID: mdl-26447940

RESUMO

With a view to identify novel and biocompatible neuroprotectants, we designed nucleoside 5'-thiophosphate analogues, 6-11. We identified 2-SMe-ADP(α-S), 7A, as a most promising neuroprotectant. 7A reduced ROS production in PC12 cells under oxidizing conditions, IC50 of 0.08 vs 21 µM for ADP. Furthermore, 7A rescued primary neurons subjected to oxidation, EC50 of 0.04 vs 19 µM for ADP. 7A is a most potent P2Y1-R agonist, EC50 of 0.0026 µM. Activity of 7A in cells involved P2Y1/12-R as indicated by blocking P2Y12-R or P2Y1-R. Compound 7A inhibited Fenton reaction better than EDTA, IC50 of 37 vs 54 µM, due to radical scavenging, IC50 of 12.5 vs 30 µM for ADP, and Fe(II)-chelation, IC50 of 80 vs >200 µM for ADP (ferrozine assay). In addition, 7A was stable in human blood serum, t1/2 of 15 vs 1.5 h for ADP, and resisted hydrolysis by NPP1/3, 2-fold vs ADP. Hence, we propose 7A as a highly promising neuroprotectant.


Assuntos
Antioxidantes/farmacologia , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Nucleosídeos/farmacologia , Nucleotídeos/farmacologia , Oligonucleotídeos Fosforotioatos/farmacologia , Animais , Antioxidantes/química , Células Cultivadas , Descoberta de Drogas , Humanos , Peróxido de Hidrogênio/antagonistas & inibidores , Hidrólise , Ferro , Modelos Moleculares , Neurônios/citologia , Neurônios/metabolismo , Fármacos Neuroprotetores/química , Nucleosídeos/química , Nucleotídeos/química , Células PC12 , Oligonucleotídeos Fosforotioatos/química , Agonistas do Receptor Purinérgico P2Y/química , Agonistas do Receptor Purinérgico P2Y/farmacologia , Ratos , Espécies Reativas de Oxigênio/metabolismo , Receptores Purinérgicos P2Y1/metabolismo
20.
Bioorg Med Chem ; 23(17): 5764-73, 2015 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-26233801

RESUMO

P2Y6 receptor (P2Y6-R) is involved in various physiological and pathophysiological events. With a view to set rules for the design of UDP-based reversible P2Y6-R antagonists as potential drugs, we established structure-activity relationship of UDP analogues, bearing modifications at the uracil ring, ribose moiety, and the phosphate chain. For instance, C5-phenyl- or 3-NMe-uridine-5'-α,ß-methylene-diphosphonate, 16 and 23, or lack of 2'-OH, in 12-15, resulted in loss of both agonist and antagonist activity toward hP2Y6-R. However, uridylyl phosphosulfate, 19, selectively inhibited hP2Y6-R (IC50 112 µM) versus P2Y2/4-Rs. In summary, we have established a comprehensive SAR for hP2Y6-R ligands towards the development of hP2Y6-R antagonists.


Assuntos
Receptores Purinérgicos P2/química , Nucleotídeos de Uracila/síntese química , Humanos , Estrutura Molecular , Relação Estrutura-Atividade , Nucleotídeos de Uracila/química
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...