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1.
Nat Chem Biol ; 18(5): 538-546, 2022 May.
Artigo em Inglês | MEDLINE | ID: mdl-35314816

RESUMO

The marine microbial natural product salinosporamide A (marizomib) is a potent proteasome inhibitor currently in clinical trials for the treatment of brain cancer. Salinosporamide A is characterized by a complex and densely functionalized γ-lactam-ß-lactone bicyclic warhead, the assembly of which has long remained a biosynthetic mystery. Here, we report an enzymatic route to the salinosporamide core catalyzed by a standalone ketosynthase (KS), SalC. Chemoenzymatic synthesis of carrier protein-tethered substrates, as well as intact proteomics, allowed us to probe the reactivity of SalC and understand its role as an intramolecular aldolase/ß-lactone synthase with roles in both transacylation and bond-forming reactions. Additionally, we present the 2.85-Å SalC crystal structure that, combined with site-directed mutagenesis, allowed us to propose a bicyclization reaction mechanism. This work challenges our current understanding of the role of KS enzymes and establishes a basis for future efforts toward streamlined production of a clinically relevant chemotherapeutic.


Assuntos
Produtos Biológicos , Lactamas , Produtos Biológicos/farmacologia , Lactonas/química , Inibidores de Proteassoma , Pirróis/farmacologia
2.
Nat Commun ; 12(1): 3038, 2021 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-34031424

RESUMO

Mayaro virus (MAYV) is an emerging arbovirus of the Americas that may cause a debilitating arthritogenic disease. The biology of MAYV is not fully understood and largely inferred from related arthritogenic alphaviruses. Here, we present the structure of MAYV at 4.4 Å resolution, obtained from a preparation of mature, infective virions. MAYV presents typical alphavirus features and organization. Interactions between viral proteins that lead to particle formation are described together with a hydrophobic pocket formed between E1 and E2 spike proteins and conformational epitopes specific of MAYV. We also describe MAYV glycosylation residues in E1 and E2 that may affect MXRA8 host receptor binding, and a molecular "handshake" between MAYV spikes formed by N262 glycosylation in adjacent E2 proteins. The structure of MAYV is suggestive of structural and functional complexity among alphaviruses, which may be targeted for specificity or antiviral activity.


Assuntos
Infecções por Alphavirus/virologia , Alphavirus/ultraestrutura , Microscopia Crioeletrônica , Espectrometria de Massas , Alphavirus/imunologia , Infecções por Alphavirus/imunologia , Animais , Anticorpos Neutralizantes , Chlorocebus aethiops , Glicosilação , Humanos , Imunoglobulinas , Proteínas de Membrana , Células Vero
3.
Eur Respir J ; 58(1)2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33361100

RESUMO

BACKGROUND: Nitazoxanide is widely available and exerts broad-spectrum antiviral activity in vitro. However, there is no evidence of its impact on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. METHODS: In a multicentre, randomised, double-blind, placebo-controlled trial, adult patients presenting up to 3 days after onset of coronavirus disease 2019 (COVID-19) symptoms (dry cough, fever and/or fatigue) were enrolled. After confirmation of SARS-CoV-2 infection using reverse transcriptase PCR on a nasopharyngeal swab, patients were randomised 1:1 to receive either nitazoxanide (500 mg) or placebo, three times daily, for 5 days. The primary outcome was complete resolution of symptoms. Secondary outcomes were viral load, laboratory tests, serum biomarkers of inflammation and hospitalisation rate. Adverse events were also assessed. RESULTS: From June 8 to August 20, 2020, 1575 patients were screened. Of these, 392 (198 placebo, 194 nitazoxanide) were analysed. Median (interquartile range) time from symptom onset to first dose of study drug was 5 (4-5) days. At the 5-day study visit, symptom resolution did not differ between the nitazoxanide and placebo arms. Swabs collected were negative for SARS-CoV-2 in 29.9% of patients in the nitazoxanide arm versus 18.2% in the placebo arm (p=0.009). Viral load was reduced after nitazoxanide compared to placebo (p=0.006). The percentage viral load reduction from onset to end of therapy was higher with nitazoxanide (55%) than placebo (45%) (p=0.013). Other secondary outcomes were not significantly different. No serious adverse events were observed. CONCLUSIONS: In patients with mild COVID-19, symptom resolution did not differ between nitazoxanide and placebo groups after 5 days of therapy. However, early nitazoxanide therapy was safe and reduced viral load significantly.


Assuntos
COVID-19 , Adulto , Humanos , Nitrocompostos , SARS-CoV-2 , Tiazóis , Resultado do Tratamento
4.
Braz J Microbiol ; 51(3): 989-997, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32333271

RESUMO

Fungi in the genus Trichoderma are notorious producers of secondary metabolites with diverse applications, such as antibacterial, antifungal, and plant growth-promoting properties. Peptaibols are linear peptides produced by such fungi, with more than 440 compounds described to date, including tricholongins, longibrachins, trichobrachins, and trichovirins. Peptaibols are synthesized by non-ribosomal peptide synthetases and they have several biological activities. Our research group isolated four peptaibols (6DP2, 6DP3, 6DP4, and 6DP5) with antifungal activity against the plant pathogen Colletotrichum gloeosporioides and the proteasome (a cancer chemotherapy target) from Trichoderma sp. P8BDA1F1, an endophytic fungus from Begonia venosa. The ethyl acetate extract of this endophyte showed activity of 6.01% and 75% against C. gloeosporioides and the proteasome, respectively. The isolated compounds were identified by MS/MS and compared to literature data, suggesting the presence of trilongins BI, BII, BIII, and BIV, which are peptaibols containing 20 amino acid residues. The minimum inhibitory concentration against C. gloeosporioides was 40 µM for trilongin BI, 320 µM for trilongin BII, 160 µM for trilongin BIII, and 310 µM for trilongin BIV. BI-BIV trilongins inhibited proteasome ChTL activity, with IC50 values of 6.5 ± 2.7; 4.7 ± 1.8; 6.3 ± 2.2; and 2.7 ± 0.5 µM, respectively. The compounds were tested ex vivo against the intracellular amastigotes of Leishmania (L.) infantum but showed no selectivity. It is the first report of trilongins BI-BIV with antifungal activity against C. gloeosporioides and the proteasome target.


Assuntos
Antifúngicos/farmacologia , Antineoplásicos/farmacologia , Begoniaceae/microbiologia , Peptaibols/farmacologia , Trichoderma/química , Antifúngicos/química , Antifúngicos/isolamento & purificação , Antineoplásicos/química , Antineoplásicos/isolamento & purificação , Colletotrichum/efeitos dos fármacos , Endófitos , Concentração Inibidora 50 , Testes de Sensibilidade Microbiana , Peptaibols/química , Peptaibols/isolamento & purificação , Filogenia , Inibidores de Proteassoma/farmacologia , Trichoderma/classificação , Trichoderma/genética , Trichoderma/isolamento & purificação
5.
Nat Prod Rep ; 37(4): 488-514, 2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-32048675

RESUMO

Covering: 2008 to 2018Over the last decade more than two hundred single natural products were confirmed as natural allosteric modulators (alloNPs) of proteins. The compounds are presented and discussed with the support of a chemical space, constructed using a principal component analysis (PCA) of molecular descriptors from chemical compounds of distinct databases. This analysis showed that alloNPs are dispersed throughout the majority of the chemical space defined by natural products in general. Moreover, a cluster of alloNPs was shown to occupy a region almost devoid of allosteric modulators retrieved from a dataset composed mainly of synthetic compounds, further highlighting the importance to explore the entire natural chemical space for probing allosteric mechanisms. The protein targets which alloNPs bind to comprised 81 different proteins, which were classified into 5 major groups, with enzymes, in particular hydrolases, being the main representative group. The review also brings a critical interpretation on the mechanisms by which alloNPs display their molecular action on proteins. In the latter analysis, alloNPs were classified according to their final effect on the target protein, resulting in 3 major categories: (i) local alteration of the orthosteric site; (ii) global alteration in protein dynamics that change function; and (iii) oligomer stabilisation or protein complex destabilisation via protein-protein interaction in sites distant from the orthosteric site. G-protein coupled receptors (GPCRs), which use a combination of the three types of allosteric regulation found, were also probed by natural products. In summary, the natural allosteric modulators reviewed herein emphasise their importance for exploring alternative chemotherapeutic strategies, potentially pushing the boundaries of the druggable space of pharmacologically relevant drug targets.


Assuntos
Regulação Alostérica/efeitos dos fármacos , Produtos Biológicos/química , Produtos Biológicos/farmacologia , Proteínas/metabolismo , Produtos Biológicos/classificação , Descoberta de Drogas/métodos , Enzimas/química , Enzimas/metabolismo , Humanos , Mapas de Interação de Proteínas , Proteínas/química , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia
6.
J Nat Prod ; 83(1): 55-65, 2020 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-31895573

RESUMO

A new method of screening was developed to generate 770 organic and water-soluble fractions from extracts of nine species of marine sponges, from the growth media of 18 species of marine-derived fungi, and from the growth media of 13 species of endophytic fungi. The screening results indicated that water-soluble fractions displayed significant bioactivity in cytotoxic, antibiotic, anti-Leishmania, anti-Trypanosoma cruzi, and inhibition of proteasome assays. Purification of water-soluble fractions from the growth medium of Penicillium solitum IS1-A provided the new glutamic acid derivatives solitumine A (1), solitumine B (2), and solitumidines A-D (3-6). The structures of compounds 1-6 have been established by analysis of spectroscopic data, chemical derivatizations, and vibrational circular dichroism calculations. Although no biological activity could be observed for compounds 1-6, the new structures reported for 1-6 indicate that the investigation of water-soluble natural products represents a relevant strategy in finding new secondary metabolites.


Assuntos
Glutamatos/química , Regiões Antárticas , Fungos/química , Estrutura Molecular , Penicillium/química , Água
8.
J Nat Prod ; 81(10): 2296-2300, 2018 10 26.
Artigo em Inglês | MEDLINE | ID: mdl-30281303

RESUMO

The new pyrrole-imidazole and pyrrole-guanidine alkaloids 4-debromooroidin (1), 4-debromougibohlin (2), 5-debromougibohlin (3), and 5-bromopalau'amine (4), along with the known hymenidin (5) and (+)-monobromoisophakellin (6), have been isolated from a Dictyonella sp. marine sponge, collected at the Amazon River mouth. The bromine-substitution pattern observed for compounds 1, 2 and 4 is unusual among bromopyrrole alkaloids isolated from marine sponges. The 20S proteasome inhibitory activities of compounds 1-6 have been recorded, with 5-bromopalau'amine (4) being the most active in this series.


Assuntos
Poríferos/química , Inibidores de Proteassoma/química , Inibidores de Proteassoma/farmacologia , Pirróis/química , Pirróis/farmacologia , Animais , Brasil , Estrutura Molecular , Complexo de Endopeptidases do Proteassoma , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética
9.
J Org Chem ; 83(9): 5160-5176, 2018 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-29644851

RESUMO

This article describes our efforts toward the total synthesis of actinoranone. Our synthesis strategies rely on a convergent route to connect the terpenoid and polyketide fragments, employing catalysis and powerful classical reactions for the assembly of these key fragments. A new transformation was disclosed during this work, a domino ring-opening and esterification. Initial cytotoxic studies for the selected synthesis intermediates are also presented.


Assuntos
Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Diterpenos/síntese química , Diterpenos/farmacologia , Antineoplásicos/química , Técnicas de Química Sintética , Diterpenos/química , Células HCT116 , Humanos , Modelos Moleculares , Conformação Molecular
10.
mSystems ; 3(2)2018.
Artigo em Inglês | MEDLINE | ID: mdl-29629418

RESUMO

Natural products are the richest source of chemical compounds for drug discovery. Particularly, bacterial secondary metabolites are in the spotlight due to advances in genome sequencing and mining, as well as for the potential of biosynthetic pathway manipulation to awake silent (cryptic) gene clusters under laboratory cultivation. Further progress in compound detection, such as the development of the tandem mass spectrometry (MS/MS) molecular networking approach, has contributed to the discovery of novel bacterial natural products. The latter can be applied directly to bacterial crude extracts for identifying and dereplicating known compounds, therefore assisting the prioritization of extracts containing novel natural products, for example. In our opinion, these three approaches-genome mining, silent pathway induction, and MS-based molecular networking-compose the tripod for modern bacterial natural product discovery and will be discussed in this perspective.

11.
Anticancer Agents Med Chem ; 17(13): 1777-1785, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28403779

RESUMO

BACKGROUND: Chronic myeloid leukemia (CML) is currently treated with imatinib, a Bcr-Abl inhibitor. However, resistance to this drug usually develops over time. Triptolide, a diterpenoid triepoxide, has been shown active against CML cells resistant to imatinib, acting mainly on the level of Bcr-Abl transcription inhibition. OBJECTIVE: Here, we used the triterpene betulinic acid, a known proteasome inhibitor with potential antileukemic activity, as a scaffold for the generation of analogues with predicted triptolide biological activity. METHOD: Betulinic acid derivatives were designed based on the structure-activity relationship of triptolide and evaluated for their cytotoxic effects in CML cells, lymphocytes and human keratinocytes (HaCaT), as well as against the proteasome complex. The main modification performed on betulinic acid was fluorination at C-28 and epoxidation, both of which are responsible for enhancing activity of triptolide. A total of 10 compounds were obtained: 6 previously described and 4 novel compounds. The cytotoxic activity over a CML cell line (K562) was assessed using flow cytometry and compared to lymphocytes and HaCaT. RESULT: The results show that betulinic acid was the most cytotoxic compound against CML cells, showing a good selectivity index for cancer over normal cells. The most important trend for the activity in betulinic acid derivatives is the presence of a free hydroxyl group at C-3 and a carboxyl group at C-28. Results also indicated that the epoxide is important for enhancing the activity, while modification at C-28 worsens the activity. CONCLUSION: Proteasome inhibition assays suggest that proteasome is the main target for betulinic acid and its derivatives.


Assuntos
Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Desenho de Fármacos , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Triterpenos/síntese química , Triterpenos/farmacologia , Antineoplásicos/uso terapêutico , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Células K562 , Triterpenos Pentacíclicos , Análise Espectral/métodos , Relação Estrutura-Atividade , Triterpenos/uso terapêutico
12.
Bioorg Med Chem ; 23(15): 4462-4471, 2015 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-26117648

RESUMO

Low molecular weight protein tyrosine phosphatases (LMW-PTP, EC 3.1.3.48) are a family of single-domain enzymes with molecular weight up to 18 kDa, expressed in different tissues and considered attractive pharmacological targets for cancer chemotherapy. Despite this, few LMW-PTP inhibitors have been described to date, and the structural information on LMW-PTP druggable binding sites is scarce. In this study, a small series of phosphonic acids were designed based on a new crystallographic structure of LMW-PTP complexed with benzylsulfonic acid, determined at 2.1Å. In silico docking was used as a tool to interpret the structural and enzyme kinetics data, as well as to design new analogs. From the synthesized series, two compounds were found to act as competitive inhibitors, with inhibition constants of 0.124 and 0.047 mM. We also report the 2.4Å structure of another complex in which LMW-PTP is bound to benzylphosphonic acid, and a structure of apo LMW-PTP determined at 2.3Å resolution. Although no appreciable conformation changes were observed, in the latter structures, amino acid residues from an expression tag were found bound to a hydrophobic region at the protein surface. This regions is neighbored by positively charged residues, adjacent to the active site pocket, suggesting that this region might be not a mere artefact of crystal contacts but an indication of a possible anchoring region for the natural substrate-which is a phosphorylated protein.


Assuntos
Ácidos Fosforosos/química , Proteínas Tirosina Fosfatases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Sítios de Ligação , Domínio Catalítico , Cristalografia por Raios X , Humanos , Cinética , Simulação de Acoplamento Molecular , Ácidos Fosforosos/metabolismo , Proteínas Tirosina Fosfatases/química , Proteínas Tirosina Fosfatases/genética , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Especificidade por Substrato , Ácidos Sulfônicos/química , Ácidos Sulfônicos/metabolismo
13.
Eur J Med Chem ; 97: 42-54, 2015 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-25938987

RESUMO

RK-682 (1) is a natural product known to selectively inhibit protein tyrosine phosphatases (PTPases) and is used commercially as a positive control for phosphatase inhibition in in vitro assays. Protein phosphatases are involved in several human diseases including diabetes, cancer and inflammation, and are considered important targets for pharmaceutical development. Here we report the synthesis of racemic RK-682 (rac-1) and a focused set of compounds, including racemic analogues of 1, dihydropyranones and C-acylated Meldrum's acid derivatives, the later obtained in one synthetic step from commercially available starting material. We further characterized the behavior of some representative compounds in aqueous solution and evaluated their in vitro PTPase binding and inhibition. Our data reveal that rac-1 and some derivatives are able to form large aggregates in solution, in which the aggregation capacity is dependent on the acyl side chain size. However, compound aggregation per se is not able to promote PTPase inhibition. Our data disclose a novel family of PTPase inhibitors (C-acylated Meldrum's acid derivatives) and that rac-1 and derivatives with an exposed latent negatively charged substructure (e.g.: the tetronic acid core of 1) can bind to the PTPase binding site, as well promiscuously to protein surfaces. The combined capacity of compounds to bind to proteins together with their intrinsic capacity to aggregate in solution seems essential to promote enzyme aggregation and thus, its inhibition. We also observed that divalent cations, such as magnesium frequently used in enzyme buffer solutions, can deplete the inhibitory activity of rac-1, thus influencing the enzyme inhibition experiment. Overall, these data help to characterize the mechanism of PTPase inhibition by rac-1 and derivatives, revealing that enzyme inhibition is not solely dependent on compound binding to the PTPase catalytic site as generally accepted in the literature. In addition, our results point to promiscuous mechanisms that influence significantly the in vitro evaluation of enzyme inhibition by rac-1. Therefore, we recommend caution when using natural or synthetic RK-682 (1) as an internal control for evaluating PTPase inhibition and selectivity, since many events can modulate the apparent enzyme inhibition.


Assuntos
Inibidores Enzimáticos/síntese química , Fosfoproteínas Fosfatases/antagonistas & inibidores , Proteínas Tirosina Fosfatases/antagonistas & inibidores , Sítios de Ligação , Domínio Catalítico , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Humanos , Concentração de Íons de Hidrogênio , Estrutura Molecular , Fosfoproteínas Fosfatases/química , Fosfoproteínas Fosfatases/farmacologia , Estereoisomerismo
14.
Chem Biol ; 21(6): 782-91, 2014 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-24930969

RESUMO

Hydroamination reactions involving the addition of an amine to an inactivated alkene are entropically prohibited and require strong chemical catalysts. While this synthetic process is efficient at generating substituted amines, there is no equivalent in small molecule-mediated enzyme inhibition. We report an unusual mechanism of proteasome inhibition that involves a hydroamination reaction of alkene derivatives of the epoxyketone natural product carmaphycin. We show that the carmaphycin enone first forms a hemiketal intermediate with the catalytic Thr1 residue of the proteasome before cyclization by an unanticipated intramolecular alkene hydroamination reaction, resulting in a stable six-membered morpholine ring. The carmaphycin enone electrophile, which does not undergo a 1,4-Michael addition as previously observed with vinyl sulfone and α,ß-unsaturated amide-based inhibitors, is partially reversible and gives insight into the design of proteasome inhibitors for cancer chemotherapy.


Assuntos
Alcenos/química , Antineoplásicos/farmacologia , Produtos Biológicos/farmacologia , Dipeptídeos/química , Cetonas/química , Peptídeos Cíclicos/química , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/farmacologia , Aminação , Antineoplásicos/síntese química , Antineoplásicos/química , Biocatálise , Produtos Biológicos/síntese química , Produtos Biológicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ciclização , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Células HCT116 , Humanos , Modelos Moleculares , Conformação Molecular , Inibidores de Proteassoma/síntese química , Inibidores de Proteassoma/química , Teoria Quântica , Relação Estrutura-Atividade
15.
J Mol Biol ; 425(16): 2878-93, 2013 Aug 23.
Artigo em Inglês | MEDLINE | ID: mdl-23707408

RESUMO

Peroxisome proliferator-activated receptors (PPARs) are members of a superfamily of nuclear transcription factors. They are involved in mediating numerous physiological effects in humans, including glucose and lipid metabolism. PPARα ligands effectively treat dyslipidemia and have significant antiinflammatory and anti-atherosclerotic activities. These effects and their ligand-dependent activity make nuclear receptors obvious targets for drug design. Here, we present the structure of the human PPARα in complex with WY14643, a member of fibrate class of drug, and a widely used PPAR activator. The crystal structure of this complex suggests that WY14643 induces activation of PPARα in an unusual bipartite mechanism involving conventional direct helix 12 stabilization and an alternative mode that involves a second ligand in the pocket. We present structural observations, molecular dynamics and activity assays that support the importance of the second site in WY14643 action. The unique binding mode of WY14643 reveals a new pattern of nuclear receptor ligand recognition and suggests a novel basis for ligand design, offering clues for improving the binding affinity and selectivity of ligand. We show that binding of WY14643 to PPARα was associated with antiinflammatory disease in a human corneal cell model, suggesting possible applications for PPARα ligands.


Assuntos
PPAR alfa/agonistas , PPAR alfa/química , Pirimidinas/química , Pirimidinas/metabolismo , Anti-Inflamatórios/química , Anti-Inflamatórios/metabolismo , Células Cultivadas , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Humanos , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Cinética , Modelos Moleculares , Simulação de Dinâmica Molecular , Conformação Proteica
16.
FEBS J ; 279(20): 3828-43, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22889056

RESUMO

Xylella fastidiosa is a Gram-negative bacterium that grows as a biofilm inside the xylem vessels of susceptible plants and causes several economically relevant crop diseases. In the present study, we report the functional and low-resolution structural characterization of the X. fastidiosa disulfide isomerase DsbC (XfDsbC). DsbC is part of the disulfide bond reduction/isomerization pathway in the bacterial periplasm and plays an important role in oxidative protein folding. In the present study, we demonstrate the presence of XfDsbC during different stages of X. fastidiosa biofilm development. XfDsbC was not detected during X. fastidiosa planktonic growth; however, after administering a sublethal copper shock, we observed an overexpression of XfDsbC that also occurred during planktonic growth. These results suggest that X. fastidiosa can use XfDsbC in vivo under oxidative stress conditions similar to those induced by copper. In addition, using dynamic light scattering and small-angle X-ray scattering, we observed that the oligomeric state of XfDsbC in vitro may be dependent on the redox environment. Under reducing conditions, XfDsbC is present as a dimer, whereas a putative tetrameric form was observed under nonreducing conditions. Taken together, our findings demonstrate the overexpression of XfDsbC during biofilm formation and provide the first structural model of a bacterial disulfide isomerase in solution.


Assuntos
Proteínas de Bactérias/química , Isomerases de Dissulfetos de Proteínas/química , Multimerização Proteica , Xylella/enzimologia , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biofilmes/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Cobre/farmacologia , Escherichia coli/enzimologia , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Teste de Complementação Genética , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Oxirredução , Doenças das Plantas/microbiologia , Isomerases de Dissulfetos de Proteínas/genética , Isomerases de Dissulfetos de Proteínas/metabolismo , Estrutura Quaternária de Proteína , Espalhamento a Baixo Ângulo , Homologia de Sequência de Aminoácidos , Difração de Raios X , Xylella/genética , Xylella/fisiologia
17.
J Struct Biol ; 180(1): 143-53, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22842046

RESUMO

Transthyretin (TTR) is a carrier protein involved in human amyloidosis. The development of small molecules that may act as TTR amyloid inhibitors is a promising strategy to treat these pathologies. Here we selected and characterized the interaction of flavonoids with the wild type and the V30M amyloidogenic mutant TTR. TTR acid aggregation was evaluated in vitro in the presence of the different flavonoids. The best TTR aggregation inhibitors were studied by Isothermal Titration Calorimetry (ITC) in order to reveal their thermodynamic signature of binding to TTRwt. Crystal structures of TTRwt in complex with the top binders were also obtained, enabling us to in depth inspect TTR interactions with these flavonoids. The results indicate that changing the number and position of hydroxyl groups attached to the flavonoid core strongly influence flavonoid recognition by TTR, either by changing ligand affinity or its mechanism of interaction with the two sites of TTR. We also compared the results obtained for TTRwt with the V30M mutant structure in the apo form, allowing us to pinpoint structural features that may facilitate or hamper ligand binding to the V30M mutant. Our data show that the TTRwt binding site is labile and, in particular, the central region of the cavity is sensible for the small differences in the ligands tested and can be influenced by the Met30 amyloidogenic mutation, therefore playing important roles in flavonoid binding affinity, mechanism and mutant protein ligand binding specificities.


Assuntos
Flavonoides/química , Pré-Albumina/química , Motivos de Aminoácidos , Sítios de Ligação , Calorimetria , Cristalografia por Raios X , Humanos , Modelos Moleculares , Mutação de Sentido Incorreto , Pré-Albumina/genética , Ligação Proteica , Multimerização Proteica , Termodinâmica
18.
Artigo em Inglês | MEDLINE | ID: mdl-22691782

RESUMO

The ORF XF2234 in the Xylella fastidiosa genome was identified as encoding a small heat-shock protein of 17.9 kDa (HSP17.9). HSP17.9 was found as one of the proteins that are induced during X. fastidiosa proliferation and infection in citrus culture. Recombinant HSP17.9 was crystallized and surface atomic force microscopy experiments were conducted with the aim of better characterizing the HSP17.9 crystals. X-ray diffraction data were collected at 2.7 Šresolution. The crystal belonged to space group P4(3)22, with unit-cell parameters a = 68.90, b = 68.90, c = 72.51 Å, and is the first small heat-shock protein to crystallize in this space group.


Assuntos
Proteínas de Choque Térmico Pequenas/química , Xylella/química , Cristalografia por Raios X , Proteínas de Choque Térmico Pequenas/ultraestrutura , Microscopia de Força Atômica
19.
PLoS One ; 7(5): e33643, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22606221

RESUMO

Peroxisome proliferator activated receptors (PPARs δ, α and γ) are closely related transcription factors that exert distinct effects on fatty acid and glucose metabolism, cardiac disease, inflammatory response and other processes. Several groups developed PPAR subtype specific modulators to trigger desirable effects of particular PPARs without harmful side effects associated with activation of other subtypes. Presently, however, many compounds that bind to one of the PPARs cross-react with others and rational strategies to obtain highly selective PPAR modulators are far from clear. GW0742 is a synthetic ligand that binds PPARδ more than 300-fold more tightly than PPARα or PPARγ but the structural basis of PPARδ:GW0742 interactions and reasons for strong selectivity are not clear. Here we report the crystal structure of the PPARδ:GW0742 complex. Comparisons of the PPARδ:GW0742 complex with published structures of PPARs in complex with α and γ selective agonists and pan agonists suggests that two residues (Val312 and Ile328) in the buried hormone binding pocket play special roles in PPARδ selective binding and experimental and computational analysis of effects of mutations in these residues confirms this and suggests that bulky substituents that line the PPARα and γ ligand binding pockets as structural barriers for GW0742 binding. This analysis suggests general strategies for selective PPARδ ligand design.


Assuntos
PPAR delta/química , PPAR delta/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Sítios de Ligação/genética , Cristalografia por Raios X , Células Hep G2 , Humanos , Ligantes , Modelos Moleculares , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , PPAR alfa/química , PPAR alfa/genética , PPAR alfa/metabolismo , PPAR delta/agonistas , PPAR delta/genética , PPAR gama/química , PPAR gama/genética , PPAR gama/metabolismo , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Tiazóis/química , Tiazóis/metabolismo
20.
J Struct Biol ; 173(2): 323-32, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-20937391

RESUMO

Transthyretin (TTR) is a tetrameric ß-sheet-rich transporter protein directly involved in human amyloid diseases. Several classes of small molecules can bind to TTR delaying its amyloid fibril formation, thus being promising drug candidates to treat TTR amyloidoses. In the present study, we characterized the interactions of the synthetic triiodo L-thyronine analogs and thyroid hormone nuclear receptor TRß-selective agonists GC-1 and GC-24 with the wild type and V30M variant of human transthyretin (TTR). To achieve this aim, we conducted in vitro TTR acid-mediated aggregation and isothermal titration calorimetry experiments and determined the TTR:GC-1 and TTR:GC-24 crystal structures. Our data indicate that both GC-1 and GC-24 bind to TTR in a non-cooperative manner and are good inhibitors of TTR aggregation, with dissociation constants for both hormone binding sites (HBS) in the low micromolar range. Analysis of the crystal structures of TTRwt:GC-1(24) complexes and their comparison with the TTRwt X-ray structure bound to its natural ligand thyroxine (T4) suggests, at the molecular level, the basis for the cooperative process displayed by T4 and the non-cooperative process provoked by both GC-1 and GC-24 during binding to TTR.


Assuntos
Pré-Albumina/química , Pré-Albumina/metabolismo , Tri-Iodotironina/química , Tri-Iodotironina/metabolismo , Acetatos/farmacologia , Compostos Benzidrílicos/farmacologia , Calorimetria , Humanos , Fenóis/farmacologia , Pré-Albumina/genética , Receptores beta dos Hormônios Tireóideos/agonistas , Difração de Raios X
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