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1.
Bioorg Med Chem ; 25(1): 327-337, 2017 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-27856239

RESUMO

We recently identified indole derivatives (IIIe and IIIf) with anti-chikungunya virus (CHIKV) activities at lower micro molar concentrations and a selective index of inhibition higher than the lead compound Arbidol. Here we highlight new structural information for the optimization of the previously identified lead compounds that contain the indole chemical core. Based on the structural data, a series of indole derivatives was synthesized and tested for their antiviral activity against chikungunya virus in Vero cell culture by a CPE reduction assay. Systematic optimization of the lead compounds resulted in tert-butyl-5-hydroxy-1-methyl-2-(2-trifluoromethysulfynyl)methyl)-indole-3-carboxylate derivative IIc with a 10-fold improved anti-CHIKV inhibitory activity (EC50=6.5±1µM) as compared to Arbidol demonstrating a potent, selective and specific inhibition of CHIKV replication with only a moderate cell protective effect against other related alphaviruses. The reported computational insights, together with the accessible synthetic procedure, pave the road towards the design of novel synthetic derivatives with enhanced anti-viral activities.


Assuntos
Antivirais/farmacologia , Vírus Chikungunya/efeitos dos fármacos , Indóis/farmacologia , Sulfóxidos/farmacologia , Replicação Viral/efeitos dos fármacos , Animais , Antivirais/síntese química , Vírus Chikungunya/fisiologia , Chlorocebus aethiops , Glicoproteínas/química , Indóis/síntese química , Simulação de Acoplamento Molecular , Células Vero , Proteínas do Envelope Viral/química
2.
Nucleic Acids Res ; 43(16): 7702-16, 2015 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-26250112

RESUMO

Many antiproliferative G-quadruplexes (G4s) arise from the folding of GT-rich strands. Among these, the Thrombin Binding Aptamer (TBA), as a rare example, adopts a monomolecular well-defined G4 structure. Nevertheless, the potential anticancer properties of TBA are severely hampered by its anticoagulant action and, consequently, no related studies have appeared so far in the literature. We wish to report here that suitable chemical modifications in the TBA sequence can preserve its antiproliferative over anticoagulant activity. Particularly, we replaced one residue of the TT or TGT loops with a dibenzyl linker to develop seven new quadruplex-forming TBA based sequences (TBA-bs), which were studied for their structural (CD, CD melting, 1D NMR) and biological (fibrinogen, PT and MTT assays) properties. The three-dimensional structures of the TBA-bs modified at T13 (TBA-bs13) or T12 (TBA-bs12), the former endowed with selective antiproliferative activity, and the latter acting as potently as TBA in both coagulation and MTT assays, were further studied by 2D NMR restrained molecular mechanics. The comparative structural analyses indicated that neither the stability, nor the topology of the G4s, but the different localization of the two benzene rings of the linker was responsible for the loss of the antithrombin activity for TBA-bs13.


Assuntos
Anticoagulantes/química , Antineoplásicos/química , Aptâmeros de Nucleotídeos/química , Anticoagulantes/farmacologia , Antineoplásicos/farmacologia , Aptâmeros de Nucleotídeos/farmacologia , Compostos de Benzil/química , Testes de Coagulação Sanguínea , Proliferação de Células/efeitos dos fármacos , Fibrinogênio , Quadruplex G , Células HeLa , Humanos , Modelos Moleculares , Desnaturação de Ácido Nucleico , Oligonucleotídeos/síntese química , Tempo de Protrombina
3.
Chembiochem ; 15(5): 652-5, 2014 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-24520055

RESUMO

In order to expand the potential applications of G-quadruplex structures, we explored the ability of heterochiral oligodeoxynucleotides based on the thrombin-binding aptamer (TBA) sequence to fold into similar complexes, with particular focus on their resistance in biological environments. A combination of CD and NMR techniques was used. Similarly to TBA, the ODN ggTTggtgtggTTgg (lower case letters indicate L residues) is able to fold into a chair-like antiparallel G-quadruplex structure, but has a slightly higher thermal stability. The discovery that heterochiral ODNs are able to form stable G-quadruplex structures opens up new possibilities for their development in several fields, as aptamers, sensors and, as recently shown, as catalysts for enantioselective reactions.


Assuntos
Aptâmeros de Nucleotídeos/química , Quadruplex G , Oligodesoxirribonucleotídeos/química , Sequência de Bases , Dicroísmo Circular , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular
4.
Chembiochem ; 15(16): 2427-34, 2014 Nov 03.
Artigo em Inglês | MEDLINE | ID: mdl-25214456

RESUMO

We report an investigation into analogues of the thrombin binding aptamer (TBA). Individual thymidines were replaced by the unusual residue 5-hydroxymethyl-2'-deoxyuridine (hmU). This differs from the canonical thymidine by a hydroxyl group on the 5-methyl group. NMR and CD data clearly indicate that all TBA derivatives retain the ability to fold into the "chair-like" quadruplex structure. The presence of the hmU residue does not significantly affect the thermal stability of the modified aptamers compared to the parent, except for analogue H9, which showed a marked increase in melting temperature. Although all TBA analogues showed decreased affinities to thrombin, H3, H7, and H9 proved to have improved anticoagulant activities. Our data open up the possibility to enhance TBA biological properties, simply by introducing small chemical modifications.


Assuntos
Anticoagulantes/química , Aptâmeros de Nucleotídeos/química , Trombina/química , Timidina/análogos & derivados , Anticoagulantes/metabolismo , Aptâmeros de Nucleotídeos/metabolismo , Sequência de Bases , Dicroísmo Circular , Fibrinogênio/química , Fibrinogênio/metabolismo , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Ligação Proteica , Trombina/metabolismo , Timidina/química
5.
Org Biomol Chem ; 12(44): 8840-3, 2014 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-25296283

RESUMO

Degradation of nucleic acids in biological environments is the major drawback of the therapeutic use of aptamers. Among the approaches used to circumvent this negative aspect, the introduction of 3'-3' inversion of polarity sites at the sequence 3'-end has successfully been proposed. However, the introduction of inversion of polarity at the ends of the sequence has never been exploited for G-quadruplex forming aptamers. In this communication we describe CD, UV, electrophoretic and biochemical investigations concerning thrombin binding aptamer analogues containing one or two inversions of polarity sites at the oligonucleotide ends. Data indicate that, in some cases, this straightforward chemical modification is able to improve, at the same time, the thermal stability, affinity to thrombin and nuclease resistance in biological environments, thus suggesting its general application as a post-SELEX modification also for other therapeutically promising aptamers adopting G-quadruplex structures.


Assuntos
Oligonucleotídeos/química , Trombina/química , Sítios de Ligação , Quadruplex G , Trombina/análogos & derivados
6.
Bioconjug Chem ; 22(7): 1309-19, 2011 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-21688831

RESUMO

Previous studies indicate that some perylene bisimide derivatives can drive the assembly of DNA G-quadruplexes, thus suggesting the possible advantage in the adoption of perylene-conjugated G-rich oligonucleotides in biological and biotechnological applications. Nevertheless, the typical poor solubility of perylene bisimides strongly limits the number of suitable chemical strategies to prepare perylene-conjugated oligonucleotides. In order to overcome these difficulties, we employed the earlier described core twisted perylene derivatives possessing unique optical and electronic properties, besides good solubility in common solvents. As a first result, the large-scale synthesis of a new dibromoperylene derivative (PEOEBr) phosphoramidite building block is herein reported. Furthermore, the structural behavior of the conjugated PEOEBr-GGGTTAGGG (HTRp2) human telomeric repeat was investigated by using CD, UV, fluorescence, and gel electrophoresis techniques in desalted water and in K(+)- and Na(+)-containing buffers. We observed that the peculiar property of PEOEBr moieties to form dimers instead of extended aggregates drives the HTRp2 strands toward dimerization and mainly promotes the formation of quadruplex species having both the 5'-ends located at the same side of the structures. However, the counterions present in solutions (K(+) or Na(+)) as well as the strand concentration, also contribute to influence the topology and the stoichiometry of formed structures. Furthermore, unlike the unmodified sequence GGGTTAGGG (HTR2), HTRp2 strands quickly associate into G-quadruplexes even in desalted water, as assessed by CD experiments.


Assuntos
Quadruplex G , Halogenação , Oligonucleotídeos/química , Compostos Organofosforados/química , Perileno/análogos & derivados , Sequência de Bases , Bromo/química , Dicroísmo Circular , Ensaio de Desvio de Mobilidade Eletroforética , Humanos , Oligonucleotídeos/síntese química , Compostos Organofosforados/síntese química , Perileno/síntese química , Espectrofotometria Ultravioleta
7.
Biochim Biophys Acta Gen Subj ; 1863(2): 351-361, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30414444

RESUMO

Some G-quadruplex (GQ) forming aptamers, such as T30695, exhibit particularly promising properties among the potential anti-HIV drugs. T30695 G-quadruplex binds to HIV-1 integrase (IN) and inhibits its activity during 3'-end processing at nanomolar concentrations. Herein we report a study concerning six T30695-GQ variants, in which the R or S chiral glycerol T, singly replaced the thymine residues at the T30695 G-quadruplex loops. CD melting, EMSA and HMRS experiments provided information about the thermal stability and the stoichiometry of T30695-GQ variants, whereas CD and 1H NMR studies were performed to evaluate the effects of the modifications on T30695-GQ topology. Furthermore, LEDGF/p75 dependent and independent integration assays were carried out to evaluate how T loop modifications impact T30695-GQ biological activities. The obtained results showed that LEDGF/p75 adversely affects the potencies of T30695 and its variants. The IN inhibitory activities of the modified aptamers also depended on the position and on the chirality (R or S) of glycerol T loop in the GQ, mostly regardless of the G-quadruplex stabilities. In view of our and literature data, we suggest that the allosteric modulation of IN tetramer conformations by LEDGF/p75 alters the interactions between the aptamers and the enzyme. Therefore, the new T30695 variants could be suitable tools in studies aimed to clarify the HIV-1 IN tetramers allostery and its role in the integration activity.


Assuntos
Aptâmeros de Nucleotídeos/farmacologia , Glicerol/farmacologia , Inibidores de Integrase de HIV/farmacologia , Integrase de HIV/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Oligonucleotídeos/farmacologia , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/genética , Quadruplex G , Variação Genética/genética , Glicerol/química , Inibidores de Integrase de HIV/química , Oligonucleotídeos/química , Oligonucleotídeos/genética , Conformação Proteica
8.
Eur J Med Chem ; 127: 715-726, 2017 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-27836196

RESUMO

Aiming to assess the biological activities of synthetic 1,4-benzoquinones, we previously synthesized different libraries of benzoquinones with lipophilic and bulky alkyl- or aryl-substituents that inhibited 5-lipoxygenase (5-LO). The high potency of 4,5-dimethoxy-3-alkyl-1,2-benzoquinones on 5-LO led to the idea to further modify the structures and thus to improve the inhibitory potential in vitro and in vivo as well as to investigate SARs. Systematic structural optimization through accurate structure-based design resulted in compound 30 (3-tridecyl-4,5-dimethoxybenzene-1,2-diol), an ubiquinol derivative that exhibited the strongest anti-inflammatory effect, with a 10-fold improved 5-LO inhibitory activity (IC50 = 28 nM) in activated neutrophils. Moreover, 30 significantly reduced inflammatory reactions in the carrageenan-induced mouse paw oedema and in zymosan-induced peritonitis in mice. Compound 30 (1 mg/kg, i.p.) potently suppressed the levels of cysteinyl-LTs 30 min after zymosan, outperforming zileuton at a dose of 10 mg/kg. The binding patterns of the quinone- and hydroquinone-based 5-LO inhibitors were analyzed by molecular docking. Together, we elucidated the optimal alkyl chain pattern of quinones and corresponding hydroquinones and reveal a series of highly potent 5-LO inhibitors with effectiveness in vivo that might be useful as anti-inflammatory drugs.


Assuntos
Araquidonato 5-Lipoxigenase/metabolismo , Benzoquinonas/química , Benzoquinonas/farmacologia , Hidroquinonas/química , Hidroquinonas/farmacologia , Inibidores de Lipoxigenase/química , Inibidores de Lipoxigenase/farmacologia , Animais , Anti-Inflamatórios/química , Anti-Inflamatórios/metabolismo , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Araquidonato 5-Lipoxigenase/química , Benzoquinonas/metabolismo , Benzoquinonas/uso terapêutico , Edema/tratamento farmacológico , Humanos , Hidroquinonas/metabolismo , Hidroquinonas/uso terapêutico , Inibidores de Lipoxigenase/metabolismo , Inibidores de Lipoxigenase/uso terapêutico , Masculino , Camundongos , Simulação de Acoplamento Molecular , Peritonite/tratamento farmacológico , Conformação Proteica , Relação Estrutura-Atividade
9.
Biochem Pharmacol ; 112: 60-71, 2016 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-27157409

RESUMO

5-Lipoxygenase (5-LO) catalyzes the first two steps in leukotriene (LT) biosynthesis. Because LTs play pivotal roles in allergy and inflammation, 5-LO represents a valuable target for anti-inflammatory drugs. Here, we investigated the molecular mechanism, the pharmacological profile, and the in vivo effectiveness of the novel 1,2-benzoquinone-featured 5-LO inhibitor RF-22c. Compound RF-22c potently inhibited 5-LO product synthesis in neutrophils and monocytes (IC50⩾22nM) and in cell-free assays (IC50⩾140nM) without affecting 12/15-LOs, cyclooxygenase (COX)-1/2, or arachidonic acid release, in a specific and reversible manner, supported by molecular docking data. Antioxidant or iron-chelating properties were not evident for RF-22c and 5-LO-regulatory cofactors like Ca(2+) mobilization, ERK-1/2 activation, and 5-LO nuclear membrane translocation and interaction with 5-LO-activating protein (FLAP) were unaffected. RF-22c (0.1mg/kg; i.p.) impaired (I) bronchoconstriction in ovalbumin-sensitized mice challenged with acetylcholine, (II) exudate formation in carrageenan-induced paw edema, and (III) zymosan-induced leukocyte infiltration in air pouches. Taken together, RF-22c is a highly selective and potent 5-LO inhibitor in intact human leukocytes with pronounced effectiveness in different models of inflammation that warrants further preclinical analysis of this agent as anti-inflammatory drug.


Assuntos
Anti-Inflamatórios/farmacologia , Araquidonato 5-Lipoxigenase/metabolismo , Benzoquinonas/farmacologia , Broncoconstrição/efeitos dos fármacos , Leucotrienos/biossíntese , Inibidores de Lipoxigenase/farmacologia , Animais , Anti-Inflamatórios/administração & dosagem , Anti-Inflamatórios/uso terapêutico , Benzoquinonas/administração & dosagem , Benzoquinonas/uso terapêutico , Plaquetas/efeitos dos fármacos , Plaquetas/enzimologia , Plaquetas/imunologia , Broncoconstrição/imunologia , Células Cultivadas , Edema/tratamento farmacológico , Edema/enzimologia , Edema/imunologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Feminino , Humanos , Inibidores de Lipoxigenase/administração & dosagem , Inibidores de Lipoxigenase/uso terapêutico , Camundongos Endogâmicos BALB C , Simulação de Acoplamento Molecular , Monócitos/efeitos dos fármacos , Monócitos/enzimologia , Monócitos/imunologia , Neutrófilos/efeitos dos fármacos , Neutrófilos/enzimologia , Neutrófilos/imunologia
10.
J Med Chem ; 55(23): 10716-28, 2012 Dec 13.
Artigo em Inglês | MEDLINE | ID: mdl-23126678

RESUMO

An acyclic pyrimidine analogue, containing a five-member cycle fused on the pyrimidine ring, was synthesized and introduced at position 7 or 12 of the 15-mer oligodeoxynucleotide GGTTGGTGTGGTTGG, known as thrombin-binding aptamer (TBA). Characterization by 1H NMR and CD spectroscopies of the resulting aptamers, TBA-T7b and TBA-T12b, showed their ability to fold into the typical antiparallel chairlike G-quadruplex structure formed by TBA. The apparent CD melting temperatures indicated that the introduction of the acyclic residue, mainly at position 7, improves the thermal stability of resulting G-quadruplexes with respect to TBA. The anticoagulant activity of the new molecules was then valued in PT assay, and it resulted that TBA-T7b is more potent than TBA in prolonging clotting time. On the other hand, in purified fibrinogen assay the thrombin inhibitory activity of both modified sequences was lower than that of TBA using human enzyme, whereas the potency trend was again reversed using bovine enzyme. Obtained structure-activity relationships were investigated by structural and computational studies. Taken together, these results reveal the active role of TBA residues T7 and T12 and the relevance of some amino acids located in the anion binding exosite I of the protein in aptamer-thrombin interaction.


Assuntos
Anticoagulantes/farmacologia , Aptâmeros de Nucleotídeos/farmacologia , Pareamento de Bases , Animais , Aptâmeros de Nucleotídeos/química , Sequência de Bases , Bovinos , Dicroísmo Circular , Humanos , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Estrutura Molecular
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