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1.
J Biol Chem ; 298(8): 102208, 2022 08.
Artigo em Inglês | MEDLINE | ID: mdl-35772496

RESUMO

Antibiotic resistance via epigenetic methylation of ribosomal RNA is one of the most prevalent strategies adopted by multidrug resistant pathogens. The erythromycin-resistance methyltransferase (Erm) methylates rRNA at the conserved A2058 position and imparts resistance to macrolides such as erythromycin. However, the precise mechanism adopted by Erm methyltransferases for locating the target base within a complicated rRNA scaffold remains unclear. Here, we show that a conserved RNA architecture, including specific bulge sites, present more than 15 Å from the reaction center, is key to methylation at the pathogenic site. Using a set of RNA sequences site-specifically labeled by fluorescent nucleotide surrogates, we show that base flipping is a prerequisite for effective methylation and that distal bases assist in the recognition and flipping at the reaction center. The Erm-RNA complex model revealed that intrinsically flipped-out bases in the RNA serve as a putative anchor point for the Erm. Molecular dynamic simulation studies demonstrated the RNA undergoes a substantial change in conformation to facilitate an effective protein-rRNA handshake. This study highlights the importance of unique architectural features exploited by RNA to impart fidelity to RNA methyltransferases via enabling allosteric crosstalk. Moreover, the distal trigger sites identified here serve as attractive hotspots for the development of combination drug therapy aimed at reversing resistance.


Assuntos
Metiltransferases , RNA Ribossômico , Antibacterianos/farmacologia , Farmacorresistência Bacteriana , Resistência Microbiana a Medicamentos/genética , Eritromicina/farmacologia , Metiltransferases/metabolismo , RNA , RNA Ribossômico/genética , RNA Ribossômico/metabolismo
2.
Nucleic Acids Res ; 49(19): e111, 2021 11 08.
Artigo em Inglês | MEDLINE | ID: mdl-34450653

RESUMO

Interconversions between nucleic acid structures play an important role in transcriptional and translational regulation and also in repair and recombination. These interconversions are frequently promoted by nucleic acid chaperone proteins. To monitor their kinetics, Förster resonance energy transfer (FRET) is widely exploited using ensemble fluorescence intensity measurements in pre-steady-state stopped-flow experiments. Such experiments only provide a weighted average of the emission of all species in solution and consume large quantities of materials. Herein, we lift these limitations by combining time-resolved fluorescence (TRF) with droplet microfluidics (DmF). We validate the innovative TRF-DmF approach by investigating the well characterized annealing of the HIV-1 (+)/(-) Primer Binding Sequences (PBS) promoted by a HIV-1 nucleocapsid peptide. Upon rapid mixing of the FRET-labelled (-)PBS with its complementary (+)PBS sequence inside microdroplets, the TRF-DmF set-up enables resolving the time evolution of sub-populations of reacting species and reveals an early intermediate with a ∼50 ps donor fluorescence lifetime never identified so far. TRF-DmF also favorably compares with single molecule experiments, as it offers an accurate control of concentrations with no upper limit, no need to graft one partner on a surface and no photobleaching issues.


Assuntos
Primers do DNA/química , HIV-1/química , Chaperonas Moleculares/química , Proteínas do Nucleocapsídeo/química , Peptídeos/química , Albumina Sérica Humana/química , Pareamento de Bases , Primers do DNA/metabolismo , Fluoresceínas/química , Fluorescência , Transferência Ressonante de Energia de Fluorescência , HIV-1/metabolismo , Humanos , Cinética , Técnicas Analíticas Microfluídicas , Chaperonas Moleculares/metabolismo , Conformação de Ácido Nucleico , Proteínas do Nucleocapsídeo/metabolismo , Peptídeos/metabolismo , Albumina Sérica Humana/metabolismo , p-Dimetilaminoazobenzeno/análogos & derivados , p-Dimetilaminoazobenzeno/química
3.
Nucleic Acids Res ; 48(16): 9218-9234, 2020 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-32797159

RESUMO

The HIV-1 Gag protein playing a key role in HIV-1 viral assembly has recently been shown to interact through its nucleocapsid domain with the ribosomal protein L7 (RPL7) that acts as a cellular co-factor promoting Gag's nucleic acid (NA) chaperone activity. To further understand how the two proteins act together, we examined their mechanism individually and in concert to promote the annealing between dTAR, the DNA version of the viral transactivation element and its complementary cTAR sequence, taken as model HIV-1 sequences. Gag alone or complexed with RPL7 was found to act as a NA chaperone that destabilizes cTAR stem-loop and promotes its annealing with dTAR through the stem ends via a two-step pathway. In contrast, RPL7 alone acts as a NA annealer that through its NA aggregating properties promotes cTAR/dTAR annealing via two parallel pathways. Remarkably, in contrast to the isolated proteins, their complex promoted efficiently the annealing of cTAR with highly stable dTAR mutants. This was confirmed by the RPL7-promoted boost of the physiologically relevant Gag-chaperoned annealing of (+)PBS RNA to the highly stable tRNALys3 primer, favoring the notion that Gag recruits RPL7 to overcome major roadblocks in viral assembly.


Assuntos
Infecções por HIV/genética , HIV-1/genética , Proteínas Ribossômicas/genética , Produtos do Gene gag do Vírus da Imunodeficiência Humana/genética , Sequência de Aminoácidos/genética , Infecções por HIV/virologia , HIV-1/patogenicidade , Interações Hospedeiro-Patógeno/genética , Humanos , Chaperonas Moleculares/genética , Conformação de Ácido Nucleico , Ácidos Nucleicos/genética , RNA Viral/genética , Montagem de Vírus/genética
4.
J Am Chem Soc ; 142(40): 16999-17014, 2020 10 07.
Artigo em Inglês | MEDLINE | ID: mdl-32915558

RESUMO

Thienoguanosine (thG) is an isomorphic guanosine (G) surrogate that almost perfectly mimics G in nucleic acids. To exploit its full potential and lay the foundation for future applications, 20 DNA duplexes, where the bases facing and neighboring thG were systematically varied, were thoroughly studied using fluorescence spectroscopy, molecular dynamics simulations, and mixed quantum mechanical/molecular mechanics calculations, yielding a comprehensive understanding of its photophysics in DNA. In matched duplexes, thG's hypochromism was larger for flanking G/C residues but its fluorescence quantum yield (QY) and lifetime values were almost independent of the flanking bases. This was attributed to high duplex stability, which maintains a steady orientation and distance between nucleobases, so that a similar charge transfer (CT) mechanism governs the photophysics of thG independently of its flanking nucleobases. thG can therefore replace any G residue in matched duplexes, while always maintaining similar photophysical features. In contrast, the local destabilization induced by a mismatch or an abasic site restores a strong dependence of thG's QY and lifetime values on its environmental context, depending on the CT route efficiency and solvent exposure of thG. Due to this exquisite sensitivity, thG appears ideal for monitoring local structural changes and single nucleotide polymorphism. Moreover, thG's dominant fluorescence lifetime in DNA is unusually long (9-29 ns), facilitating its selective measurement in complex media using a lifetime-based or a time-gated detection scheme. Taken together, our data highlight thG as an outstanding emissive substitute for G with good QY, long fluorescence lifetimes, and exquisite sensitivity to local structural changes.


Assuntos
Sondas de DNA/química , DNA/química , Corantes Fluorescentes/química , Guanosina/análogos & derivados , Guanosina/química , Cinética , Simulação de Dinâmica Molecular , Conformação de Ácido Nucleico , Solventes/química , Espectrometria de Fluorescência , Relação Estrutura-Atividade
5.
Chemistry ; 25(58): 13363-13375, 2019 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-31322780

RESUMO

During DNA replication, ubiquitin-like, containing PHD and RING fingers domains 1 (UHRF1) plays key roles in the inheritance of methylation patterns to daughter strands by recognizing through its SET and RING-associated domain (SRA) the methylated CpGs and recruiting DNA methyltransferase 1 (DNMT1). Herein, our goal is to identify UHRF1 inhibitors targeting the 5'-methylcytosine (5mC) binding pocket of the SRA domain to prevent the recognition and flipping of 5mC and determine the molecular and cellular consequences of this inhibition. For this, we used a multidisciplinary strategy combining virtual screening and molecular modeling with biophysical assays in solution and cells. We identified an anthraquinone compound able to bind to the 5mC binding pocket and inhibit the base-flipping process in the low micromolar range. We also showed in cells that this hit impaired the UHRF1/DNMT1 interaction and decreased the overall methylation of DNA, highlighting the critical role of base flipping for DNMT1 recruitment and providing the first proof of concept of the druggability of the 5mC binding pocket. The selected anthraquinone appears thus as a key tool to investigate the role of UHRF1 in the inheritance of methylation patterns, as well as a starting point for hit-to-lead optimizations.


Assuntos
Antraquinonas/química , Proteínas Estimuladoras de Ligação a CCAAT/antagonistas & inibidores , Inibidores Enzimáticos/química , 5-Metilcitosina/química , Sítios de Ligação , DNA (Citosina-5-)-Metiltransferase 1/química , Avaliação Pré-Clínica de Medicamentos/métodos , Células HeLa , Humanos , Cinética , Metilação , Simulação de Acoplamento Molecular , Estrutura Molecular , Ligação Proteica , Conformação Proteica , Relação Estrutura-Atividade , Transfecção/métodos , Ubiquitina-Proteína Ligases
6.
Chemistry ; 24(52): 13850-13861, 2018 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-29989220

RESUMO

Nucleic acids are characterized by a variety of dynamically interconverting structures that play a major role in transcriptional and translational regulation as well as recombination and repair. To monitor these interconversions, Förster resonance energy transfer (FRET)-based techniques can be used, but require two fluorophores that are typically large and can alter the DNA/RNA structure and protein binding. Additionally, events that do not alter the donor/acceptor distance and/or angular relationship are frequently left undetected. A more benign approach relies on fluorescent nucleobases that can substitute their native counterparts with minimal perturbation, such as the recently developed 2-thienyl-3-hydroxychromone (3HCnt) and thienoguanosine (th G). To demonstrate the potency of 3HCnt and th G in deciphering interconversion mechanisms, we used the conversion of the (-)DNA copy of the HIV-1 primer binding site (-)PBS stem-loop into (+)/(-)PBS duplex, as a model system. When incorporated into the (-)PBS loop, the two probes were found to be highly sensitive to the individual steps both in the absence and the presence of a nucleic acid chaperone, providing the first complete mechanistic description of this critical process in HIV-1 replication. The combination of the two distinct probes appears to be instrumental for characterizing structural transitions of nucleic acids under various stimuli.


Assuntos
Transferência Ressonante de Energia de Fluorescência/métodos , Ácidos Nucleicos/química , Nucleosídeos/química , Sítios de Ligação , Corantes Fluorescentes/química , Cinética , Conformação de Ácido Nucleico , Ligação Proteica , Conformação Proteica , Proteínas/química , Termodinâmica
7.
J Biol Chem ; 291(10): 5116-27, 2016 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-26792864

RESUMO

The low density lipoprotein receptor-related protein 1 (LRP1) is a ubiquitously expressed cell surface receptor that protects from intracellular cholesterol accumulation. However, the underlying mechanisms are unknown. Here we show that the extracellular (α) chain of LRP1 mediates TGFß-induced enhancement of Wnt5a, which limits intracellular cholesterol accumulation by inhibiting cholesterol biosynthesis and by promoting cholesterol export. Moreover, we demonstrate that the cytoplasmic (ß) chain of LRP1 suffices to limit cholesterol accumulation in LRP1(-/-) cells. Through binding of Erk2 to the second of its carboxyl-terminal NPXY motifs, LRP1 ß-chain positively regulates the expression of ATP binding cassette transporter A1 (ABCA1) and of neutral cholesterol ester hydrolase (NCEH1). These results highlight the unexpected functions of LRP1 and the canonical Wnt5a pathway and new therapeutic potential in cholesterol-associated disorders including cardiovascular diseases.


Assuntos
Colesterol/metabolismo , Receptores de LDL/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Via de Sinalização Wnt , Transportador 1 de Cassete de Ligação de ATP/metabolismo , Sequência de Aminoácidos , Animais , Células HEK293 , Humanos , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Receptores de LDL/química , Receptores de LDL/genética , Esterol Esterase/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Proteínas Supressoras de Tumor/química , Proteínas Supressoras de Tumor/genética , Proteínas Wnt/metabolismo , Proteína Wnt-5a
8.
J Am Chem Soc ; 139(6): 2520-2528, 2017 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-28112929

RESUMO

DNA methylation patterns, which are critical for gene expression, are replicated by DNA methyltransferase 1 (DNMT1) and ubiquitin-like containing PHD and RING finger domains 1 (UHRF1) proteins. This replication is initiated by the recognition of hemimethylated CpG sites and further flipping of methylated cytosines (mC) by the Set and Ring Associated (SRA) domain of UHRF1. Although crystallography has shed light on the mechanism of mC flipping by SRA, tools are required to monitor in real time how SRA reads DNA and flips the modified nucleobase. To accomplish this aim, we have utilized two distinct fluorescent nucleobase surrogates, 2-thienyl-3-hydroxychromone nucleoside (3HCnt) and thienoguanosine (thG), incorporated at different positions into hemimethylated (HM) and nonmethylated (NM) DNA duplexes. Large fluorescence changes were associated with mC flipping in HM duplexes, showing the outstanding sensitivity of both nucleobase surrogates to the small structural changes accompanying base flipping. Importantly, the nucleobase surrogates marginally affected the structure of the duplex and its affinity for SRA at positions where they were responsive to base flipping, illustrating their promise as nonperturbing probes for monitoring such events. Stopped-flow studies using these two distinct tools revealed the fast kinetics of SRA binding and sliding to NM duplexes, consistent with its reader role. In contrast, the kinetics of mC flipping was found to be much slower in HM duplexes, substantially increasing the lifetime of CpG-bound UHRF1, and thus the probability of recruiting DNMT1 to faithfully duplicate the DNA methylation profile. The fluorescence-based approach using these two different fluorescent nucleoside surrogates advances the mechanistic understanding of the UHRF1/DNMT1 tandem and the development of assays for the identification of base flipping inhibitors.


Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Citosina/metabolismo , DNA/metabolismo , Termodinâmica , Proteínas Estimuladoras de Ligação a CCAAT/química , Citosina/química , DNA/química , Metilação de DNA , Replicação do DNA , Fluorescência , Humanos , Cinética , Estrutura Molecular , Ubiquitina-Proteína Ligases
9.
Nucleic Acids Res ; 42(2): 1065-78, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24153111

RESUMO

The HIV-1 transactivator of transcription (Tat) protein is thought to stimulate reverse transcription (RTion). The Tat protein and, more specifically, its (44-61) domain were recently shown to promote the annealing of complementary DNA sequences representing the HIV-1 transactivation response element TAR, named dTAR and cTAR, that plays a key role in RTion. Moreover, the kinetic mechanism of the basic Tat(44-61) peptide in this annealing further revealed that this peptide constitutes a representative nucleic acid annealer. To further understand the structure-activity relationships of this highly conserved domain, we investigated by electrophoresis and fluorescence approaches the binding and annealing properties of various Tat(44-61) mutants. Our data showed that the Tyr47 and basic residues of the Tat(44-61) domain were instrumental for binding to cTAR through stacking and electrostatic interactions, respectively, and promoting its annealing with dTAR. Furthermore, the annealing efficiency of the mutants clearly correlates with their ability to rapidly associate and dissociate the complementary oligonucleotides and to promote RTion. Thus, transient and dynamic nucleic acid interactions likely constitute a key mechanistic component of annealers and the role of Tat in the late steps of RTion. Finally, our data suggest that Lys50 and Lys51 acetylation regulates Tat activity in RTion.


Assuntos
Repetição Terminal Longa de HIV , HIV-1 , Transcrição Reversa , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Oligonucleotídeos/química , Peptídeos/química , Peptídeos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Produtos do Gene tat do Vírus da Imunodeficiência Humana/química
10.
J Biol Chem ; 288(36): 25895-25907, 2013 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-23843463

RESUMO

The antithrombin (AT) binding properties of heparin and low molecular weight heparins are strongly associated to the presence of the pentasaccharide sequence AGA*IA (A(NAc,6S)-GlcUA-A(NS,3,6S)-I(2S)-A(NS,6S)). By using the highly chemoselective depolymerization to prepare new ultra low molecular weight heparin and coupling it with the original separation techniques, it was possible to isolate a polysaccharide with a biosynthetically unexpected structure and excellent antithrombotic properties. It consisted of a dodecasaccharide containing an unsaturated uronate unit at the nonreducing end and two contiguous AT-binding sequences separated by a nonsulfated iduronate residue. This novel oligosaccharide was characterized by NMR spectroscopy, and its binding with AT was determined by fluorescence titration, NMR, and LC-MS. The dodecasaccharide displayed a significantly increased anti-FXa activity compared with those of the pentasaccharide, fondaparinux, and low molecular weight heparin enoxaparin.


Assuntos
Fator Xa/química , Fibrinolíticos , Oligossacarídeos , Sequência de Carboidratos , Fibrinolíticos/síntese química , Fibrinolíticos/química , Heparina , Humanos , Espectroscopia de Ressonância Magnética , Oligossacarídeos/síntese química , Oligossacarídeos/química
11.
Biochem J ; 449(2): 343-51, 2013 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-23083208

RESUMO

The 3-O-sulfation of N-sulfated glucosamine is the last event in the biosynthesis of heparin/heparan sulfate, giving rise to the antithrombin-binding pentasaccharide sequence AGA*IA, which is largely associated with the antithrombotic activity of these molecules. The aim of the present study was the structural and biochemical characterization of a previously unreported AGA*IA*-containing octasaccharide isolated from the very-low-molecular-mass heparin semuloparin, in which both glucosamine residues of the pentasaccharide moiety located at the non-reducing end bear 3-O-sulfate groups. Two-dimensional and STD (saturation transfer difference) NMR experiments clearly confirmed its structure and identified its ligand epitope binding to antithrombin. The molecular conformation of the octasaccharide-antithrombin complex has been determined by NMR experiments and docking/energy minimization. The presence of the second 3-O-sulfated glucosamine in the octasaccharide induced more than one order of magnitude increase in affinity to antithrombin compared to the pentasaccharide AGA*IA.


Assuntos
Antitrombinas/química , Glucosamina/química , Heparina/química , Oligossacarídeos/química , Antitrombinas/metabolismo , Sequência de Carboidratos , Glucosamina/metabolismo , Heparina/metabolismo , Cinética , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Conformação Molecular , Dados de Sequência Molecular , Estrutura Molecular , Oligossacarídeos/metabolismo , Ligação Proteica , Conformação Proteica , Sulfatos/química , Sulfatos/metabolismo , Temperatura
12.
Nucleic Acids Res ; 39(9): 3903-16, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21227929

RESUMO

An essential step of the reverse transcription of the HIV-1 genome is the first strand transfer that requires the annealing of the TAR RNA hairpin to the cTAR DNA hairpin. HIV-1 nucleocapsid protein (NC) plays a crucial role by facilitating annealing of the complementary hairpins. Using nuclear magnetic resonance and gel retardation assays, we investigated the interaction between NC and the top half of the cTAR DNA (mini-cTAR). We show that NC(11-55) binds the TGG sequence in the lower stem that is destabilized by the adjacent internal loop. The 5' thymine interacts with residues of the N-terminal zinc knuckle and the 3' guanine is inserted in the hydrophobic plateau of the C-terminal zinc knuckle. The TGG sequence is preferred relative to the apical and internal loops containing unpaired guanines. Investigation of the DNA-protein contacts shows the major role of hydrophobic interactions involving nucleobases and deoxyribose sugars. A similar network of hydrophobic contacts is observed in the published NC:DNA complexes, whereas NC contacts ribose differently in NC:RNA complexes. We propose that the binding polarity of NC is related to these contacts that could be responsible for the preferential binding to single-stranded nucleic acids.


Assuntos
DNA Viral/química , Desoxirribose/química , Repetição Terminal Longa de HIV , Produtos do Gene gag do Vírus da Imunodeficiência Humana/química , Sequência de Aminoácidos , DNA Viral/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismo
13.
Antimicrob Agents Chemother ; 56(2): 1010-8, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22083480

RESUMO

Since currently available therapies against HIV/AIDS still show important drawbacks, the development of novel anti-HIV treatments is a key issue. We recently characterized methylated oligoribonucleotides (mONs) that extensively inhibit HIV-1 replication in primary T cells at nanomolar concentrations. The mONs were shown to target both HIV-1 reverse transcriptase (RT) and the nucleocapsid protein (NC), which is an essential partner of RT during viral DNA synthesis. To further understand the mechanism of such mONs, we studied by isothermal titration calorimetry and fluorescence-based techniques their NC binding properties and ability to inhibit the nucleic acid chaperone properties of NC. Notably, we investigated the ability of mONs to inhibit the NC-induced destabilization of the HIV-1 cTAR (complementary DNA sequence to TAR [transactivation response element]) stem-loop and the NC-promoted cTAR annealing to its complementary sequence, required at the early stage of HIV-1 viral DNA synthesis. Moreover, we compared the activity of the mONs to that of a number of modified and nonmodified oligonucleotides. Results show that the mONs inhibit NC by a competitive mechanism whereby the mONs tightly bind the NC peptide, mainly through nonelectrostatic interactions with the hydrophobic platform at the top of the NC zinc fingers. Taken together, these results favor the notion that the mONs impair the process of the RT-directed viral DNA synthesis by sequestering NC molecules, thus preventing the chaperoning of viral DNA synthesis by NC. These findings contribute to the understanding of the molecular basis for NC inhibition by mONs, which could be used for the rational design of antiretroviral compounds targeting HIV-1 NC protein.


Assuntos
HIV-1/metabolismo , Chaperonas Moleculares/antagonistas & inibidores , Proteínas do Nucleocapsídeo/antagonistas & inibidores , Oligorribonucleotídeos/farmacologia , Sequência de Aminoácidos , Sequência de Bases , DNA Viral/biossíntese , Transcriptase Reversa do HIV/antagonistas & inibidores , Humanos , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Proteínas do Nucleocapsídeo/química , Proteínas do Nucleocapsídeo/metabolismo , Oligorribonucleotídeos/química , Oligorribonucleotídeos/metabolismo
14.
ACS Med Chem Lett ; 11(5): 698-705, 2020 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-32435373

RESUMO

The HIV-1 nucleocapsid protein 7 (NC) is a potential target for effective antiretroviral therapy due to its central role in virus replication, mainly linked to nucleic acid (NA) chaperone activity, and low susceptibility to drug resistance. By screening a compounds library, we identified the aminopyrrolic compound CN14_17, a known carbohydrate binding agent, that inhibits the NC chaperone activity in the low micromolar range. Different from most of available NC inhibitors, CN14_17 fully prevents the NC-induced annealing of complementary NA sequences. Using fluorescence assays and isothermal titration calorimetry, we found that CN14_17 competes with NC for the binding to NAs, preferentially targeting single-stranded sequences. Molecular dynamics simulations confirmed that binding to cTAR occurs preferably within the guanosine-rich single stranded sequence. Finally, CN14_17 exhibited antiretroviral activity in the low micromolar range, although with a moderate therapeutic index. Overall, CN14_17 might be the progenitor of a new promising class of NC inhibitors.

15.
ACS Infect Dis ; 6(4): 687-702, 2020 04 10.
Artigo em Inglês | MEDLINE | ID: mdl-32045204

RESUMO

The nucleocapsid protein (NC) is a highly conserved protein that plays key roles in HIV-1 replication through its nucleic acid chaperone properties mediated by its two zinc fingers and basic residues. NC is a promising target for antiviral therapy, particularly to control viral strains resistant to currently available drugs. Since calixarenes with antiviral properties have been described, we explored the ability of calixarene hydroxymethylphosphonic or sulfonic acids to inhibit NC chaperone properties and exhibit antiviral activity. By using fluorescence-based assays, we selected four calixarenes inhibiting NC chaperone activity with submicromolar IC50 values. These compounds were further shown by mass spectrometry, isothermal titration calorimetry, and fluorescence anisotropy to bind NC with no zinc ejection and to compete with nucleic acids for the binding to NC. Molecular dynamic simulations further indicated that these compounds interact via their phosphonate or sulfonate groups with the basic surface of NC but not with the hydrophobic plateau at the top of the folded fingers. Cellular studies showed that the most soluble compound CIP201 inhibited the infectivity of wild-type and drug-resistant HIV-1 strains at low micromolar concentrations, primarily targeting the early steps of HIV-1 replication. Moreover, CIP201 was also found to inhibit the flipping and polymerization activity of reverse transcriptase. Calixarenes thus form a class of noncovalent NC inhibitors, endowed with a new binding mode and multitarget antiviral activity.


Assuntos
Fármacos Anti-HIV/farmacologia , Calixarenos/farmacologia , HIV-1/química , Chaperonas Moleculares/antagonistas & inibidores , Proteínas do Nucleocapsídeo/antagonistas & inibidores , Organofosfonatos/farmacologia , Animais , Calixarenos/classificação , HIV-1/efeitos dos fármacos , Concentração Inibidora 50 , Camundongos , Camundongos Transgênicos , Simulação de Dinâmica Molecular , Ligação Proteica
16.
Biochem J ; 400(3): 467-76, 2006 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-16839309

RESUMO

We have purified to homogeneity two forms of a new serine protease inhibitor specific for elastase/chymotrypsin from the ovary gland of the desert locust Schistocerca gregaria. This protein, greglin, has 83 amino acid residues and bears putative phosphorylation sites. Amino acid sequence alignments revealed no homology with pacifastin insect inhibitors and only a distant relationship with Kazal-type inhibitors. This was confirmed by computer-based structural studies. The most closely related homologue is a putative gene product from Ciona intestinalis with which it shares 38% sequence homology. Greglin is a fast-acting and tight binding inhibitor of human neutrophil elastase (k(ass)=1.2x10(7) M(-1) x s(-1), K(i)=3.6 nM) and subtilisin. It also binds neutrophil cathepsin G, pancreatic elastase and chymotrypsin with a lower affinity (26 nM< or =K(i)< or =153 nM), but does not inhibit neutrophil protease 3 or pancreatic trypsin. The capacity of greglin to inhibit neutrophil elastase was not significantly affected by exposure to acetonitrile, high temperature (90 degrees C), low or high pH (2.5-11.0), N-chlorosuccinimide-mediated oxidation or the proteolytic enzymes trypsin, papain and pseudolysin from Pseudomonas aeruginosa. Greglin efficiently inhibits the neutrophil elastase activity of sputum supernatants from cystic fibrosis patients. Its biological function in the locust ovary gland is currently unknown, but its physicochemical properties suggest that it can be used as a template to design a new generation of highly resistant elastase inhibitors for treating inflammatory diseases.


Assuntos
Gafanhotos/química , Elastase de Leucócito/antagonistas & inibidores , Elastase de Leucócito/metabolismo , Inibidores de Serina Proteinase/metabolismo , Inibidores de Serina Proteinase/farmacologia , Sequência de Aminoácidos , Animais , Feminino , Modelos Moleculares , Ovário/metabolismo , Conformação Proteica , Isoformas de Proteínas , Inibidores de Serina Proteinase/química
17.
FEBS J ; 272(22): 5883-93, 2005 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-16279952

RESUMO

Neutrophil proteinase-mediated lung tissue destruction is prevented by inhibitors, including elafin and its precursor, trappin. We wanted to establish whether neutrophil-derived oxidants might impair the inhibitory function of these molecules. Myeloperoxidase/H(2)O(2) and N-chlorosuccinimide oxidation of the inhibitors was checked by mass spectrometry and enzymatic methods. Oxidation significantly lowers the affinities of the two inhibitors for neutrophil elastase (NE) and proteinase 3 (Pr3). This decrease in affinity is essentially caused by an increase in the rate of inhibitory complex dissociation. Oxidized elafin and trappin have, however, reasonable affinities for NE (K(i) = 4.0-9.2 x 10(-9) M) and for Pr3 (K(i) = 2.5-5.0 x 10(-8) M). These affinities are theoretically sufficient to allow the oxidized inhibitors to form tight binding complexes with NE and Pr3 in lung secretions where their physiological concentrations are in the micromolar range. Yet, they are unable to efficiently inhibit the elastolytic activity of the two enzymes. At their physiological concentration, fully oxidized elafin and trappin do not inhibit more than 30% of an equimolar concentration of NE or Pr3. We conclude that in vivo oxidation of elafin and trappin strongly impairs their activity. Inhibitor-based therapy of inflammatory lung diseases must be carried out using oxidation-resistant variants of these molecules.


Assuntos
Inibidores Enzimáticos/farmacologia , Precursores Enzimáticos/farmacologia , Elastase de Leucócito/antagonistas & inibidores , Proteínas/farmacologia , Serina Endopeptidases/metabolismo , Elafina , Inibidores Enzimáticos/química , Precursores Enzimáticos/química , Precursores Enzimáticos/genética , Variação Genética , Humanos , Cinética , Elastase de Leucócito/metabolismo , Espectrometria de Massas , Metionina/metabolismo , Mieloblastina , Oxirredução , Precursores de Proteínas/antagonistas & inibidores , Precursores de Proteínas/metabolismo , Proteínas Secretadas Inibidoras de Proteinases , Proteínas/química , Proteínas/genética , Proteínas Recombinantes/biossíntese
18.
J Mol Biol ; 427(6 Pt B): 1480-1494, 2015 Mar 27.
Artigo em Inglês | MEDLINE | ID: mdl-25644662

RESUMO

The Pr55 Gag of human immunodeficiency virus type 1 orchestrates viral particle assembly in producer cells, which requires the genomic RNA and a lipid membrane as scaffolding platforms. The nucleocapsid (NC) domain with its two invariant CCHC zinc fingers flanked by unfolded basic sequences is thought to direct genomic RNA selection, dimerization and packaging during virus assembly. To further investigate the role of NC domain, we analyzed the assembly of Gag with deletions in the NC domain in parallel with that of wild-type Gag using fluorescence lifetime imaging microscopy combined with Förster resonance energy transfer in HeLa cells. We found that, upon binding to nucleic acids, the NC domain promotes the formation of compact Gag oligomers in the cytoplasm. Moreover, the intracellular distribution of the population of oligomers further suggests that oligomers progressively assemble during their trafficking toward the plasma membrane (PM), but with no dramatic changes in their compact arrangement. This ultimately results in the accumulation at the PM of closely packed Gag oligomers that likely arrange in hexameric lattices, as revealed by the perfect match between the experimental Förster resonance energy transfer value and the one calculated from the structural model of Gag in immature viruses. The distal finger and flanking basic sequences, but not the proximal finger, appear to be essential for Gag oligomer compaction and membrane binding. Moreover, the full NC domain was found to be instrumental in the kinetics of Gag oligomerization and intracellular trafficking. These findings further highlight the key roles played by the NC domain in virus assembly.


Assuntos
Membrana Celular/metabolismo , Infecções por HIV/metabolismo , Microscopia de Fluorescência , Dedos de Zinco/genética , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismo , Citoplasma/metabolismo , Transferência Ressonante de Energia de Fluorescência , Infecções por HIV/virologia , HIV-1/fisiologia , Humanos , Mutação/genética , Nucleocapsídeo , Ligação Proteica , Multimerização Proteica , Transporte Proteico , RNA Viral/genética , Montagem de Vírus , Produtos do Gene gag do Vírus da Imunodeficiência Humana/química , Produtos do Gene gag do Vírus da Imunodeficiência Humana/genética
19.
Lab Chip ; 14(10): 1767-74, 2014 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-24683603

RESUMO

We developed a new experimental approach combining Time-Resolved Fluorescence (TRF) spectroscopy and Droplet Microfluidics (DµF) to investigate the relaxation dynamics of structurally heterogeneous biomolecular systems. Here DµF was used to produce with minimal material consumption an out-of-equilibrium, fluorescently labeled biomolecular complex by rapid mixing within the droplets. TRF detection was implemented with a streak camera to monitor the time evolution of the structural heterogeneity of the complex along its relaxation towards equilibrium while it propagates inside the microfluidic channel. The approach was validated by investigating the fluorescence decay kinetics of a model interacting system of bovine serum albumin and Patent Blue V. Fluorescence decay kinetics are acquired with very good signal-to-noise ratio and allow for global, multicomponent fluorescence decay analysis, evidencing heterogeneous structural relaxation over several 100 ms.


Assuntos
Corantes Fluorescentes/química , Técnicas Analíticas Microfluídicas/métodos , Corantes de Rosanilina/química , Soroalbumina Bovina/química , Espectrometria de Fluorescência , Animais , Bovinos , Cinética , Técnicas Analíticas Microfluídicas/instrumentação , Óleos/química , Soroalbumina Bovina/metabolismo , Razão Sinal-Ruído , Fatores de Tempo , Água/química
20.
ACS Chem Biol ; 9(9): 1950-5, 2014 Sep 19.
Artigo em Inglês | MEDLINE | ID: mdl-24988251

RESUMO

The nucleocapsid protein (NC) is a highly conserved protein in diverse HIV-1 subtypes that plays a central role in virus replication, mainly by interacting with conserved nucleic acid sequences. NC is considered a highly profitable drug target to inhibit multiple steps in the HIV-1 life cycle with just one compound, a unique property not shown by any of the other antiretroviral classes. However, most of NC inhibitors developed so far act through an unspecific and potentially toxic mechanism (zinc ejection) and are mainly being investigated as topical microbicides. In an effort to provide specific NC inhibitors that compete for the binding of nucleic acids to NC, here we combined molecular modeling, organic synthesis, biophysical studies, NMR spectroscopy, and antiviral assays to design, synthesize, and characterize an efficient NC inhibitor endowed with antiviral activity in vitro, a desirable property for the development of efficient antiretroviral lead compounds.


Assuntos
Fármacos Anti-HIV/química , Fármacos Anti-HIV/farmacologia , Proteínas do Nucleocapsídeo/antagonistas & inibidores , Fármacos Anti-HIV/síntese química , Calorimetria/métodos , Técnicas de Química Sintética , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos/métodos , HIV-1/química , HIV-1/efeitos dos fármacos , Células HeLa/efeitos dos fármacos , Células HeLa/virologia , Humanos , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Simulação de Acoplamento Molecular , Proteínas do Nucleocapsídeo/metabolismo , Relação Estrutura-Atividade , Tiazóis/química
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