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/farmacologiaRESUMO
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 3â 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 (500â mg) or placebo, three times daily, for 5â 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)â 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 5â 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 TratamentoRESUMO
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/farmacologiaRESUMO
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 , ÁguaRESUMO
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 MolecularRESUMO
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/genéticaRESUMO
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/metabolismoRESUMO
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 , TermodinâmicaRESUMO
The ORF XF2234 in the Xylella fastidiosa genome was identified as encoding a small heat-shock protein of 17.9â 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 2.7â Å resolution. 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 AtômicaRESUMO
The ongoing COVID-19 pandemic caused a significant loss of human lives and a worldwide decline in quality of life. Treatment of COVID-19 patients is challenging, and specific treatments to reduce COVID-19 aggravation and mortality are still necessary. Here, we describe the discovery of a novel class of epiandrosterone steroidal compounds with cationic amphiphilic properties that present antiviral activity against SARS-CoV-2 in the low micromolar range. Compounds were identified in screening campaigns using a cytopathic effect-based assay in Vero CCL81 cells, followed by hit compound validation and characterization. Compounds LNB167 and LNB169 were selected due to their ability to reduce the levels of infectious viral progeny and viral RNA levels in Vero CCL81, HEK293, and HuH7.5 cell lines. Mechanistic studies in Vero CCL81 cells indicated that LNB167 and LNB169 inhibited the initial phase of viral replication through mechanisms involving modulation of membrane lipids and cholesterol in host cells. Selection of viral variants resistant to steroidal compound treatment revealed single mutations on transmembrane, lipid membrane-interacting Spike and Envelope proteins. Finally, in vivo testing using the hACE2 transgenic mouse model indicated that SARS-CoV-2 infection could not be ameliorated by LNB167 treatment. We conclude that anti-SARS-CoV-2 activities of steroidal compounds LNB167 and LNB169 are likely host-targeted, consistent with the properties of cationic amphiphilic compounds that modulate host cell lipid biology. Although effective in vitro, protective effects were cell-type specific and did not translate to protection in vivo, indicating that subversion of lipid membrane physiology is an important, yet complex mechanism involved in SARS-CoV-2 replication and pathogenesis.
Assuntos
Tratamento Farmacológico da COVID-19 , SARS-CoV-2 , Animais , Antivirais/farmacologia , Chlorocebus aethiops , Células HEK293 , Humanos , Lipídeos , Camundongos , Pandemias , Qualidade de Vida , Células Vero , Replicação ViralRESUMO
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 XRESUMO
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 VeroRESUMO
Transthyretin (TTR) is a tetrameric beta-sheet-rich transporter protein directly involved in human amyloid diseases. It was recently found that the isoflavone genistein (GEN) potently inhibits TTR amyloid fibril formation (Green et al., 2005) and is therefore a promising candidate for TTR amyloidosis treatment. Here we used structural and biophysical approaches to characterize genistein binding to the wild type (TTRwt) and to its most frequent amyloidogenic variant, the V30M mutant. In a dose-dependent manner, genistein elicited considerable increases in both mutant and TTRwt stability as demonstrated by high hydrostatic pressure (HHP) and acid-mediated dissociation/denaturation assays. TTR:GEN crystal complexes and isothermal titration calorimetry (ITC) experiments showed that the binding mechanisms of genistein to the TTRwt and to V30M are different and are dependent on apoTTR structure conformations. Furthermore, we could also identify potential allosteric movements caused by genistein binding to the wild type TTR that explains, at least in part, the frequently observed negatively cooperative process between the two sites of TTRwt when binding ligands. These findings show that TTR mutants may present different ligand recognition and therefore are of value in ligand design for inhibiting TTR amyloidosis.
Assuntos
Amiloide/química , Amiloide/metabolismo , Genisteína/metabolismo , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Pré-Albumina/química , Pré-Albumina/metabolismo , Sítio Alostérico , Substituição de Aminoácidos , Amiloide/genética , Amiloidose/etiologia , Amiloidose/metabolismo , Cristalografia por Raios X , Humanos , Concentração de Íons de Hidrogênio , Pressão Hidrostática , Técnicas In Vitro , Ligantes , Modelos Moleculares , Proteínas Mutantes/genética , Pré-Albumina/genética , Ligação Proteica , Conformação Proteica , Estabilidade Proteica , Estrutura Quaternária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , TermodinâmicaRESUMO
Joinville is an important industrial city in Santa Catarina, southern Brazil, and also a risk factor for the Babitonga drainage basin. Oxidative stress-related parameters were evaluated in caged tilapia (Oreochromis niloticus) exposed for 7 days (sites S1 and S2) in a Babitonga drainage basin tributary river. Site S1 showed enhanced levels of hepatic CYP1A, CYP2B-like and glutathione S-transferase activity, while site S2 showed decreased levels of glutathione and increased lipoperoxidation indexes, catalase, glutathione peroxidase and glutathione reductase activity. Correlation analyses revealed that oxidative stress-related parameters behaved like a group of interrelated variables, while CYPs and glutathione S-transferase seem to be independent. New putative biomarkers were evaluated in the tilapia brain. Caspase-3 activation (both sites), decreased in p38MAPK phosphorylation (site S2) and decreased expression in HSP70 (site S1) were observed. Data indicate that employed variables, when used as a group (oxidative stress-related parameters, CYP1A/2B-like, caspase-3, HSP70 and protein kinases) can be useful as predictors of pollution.
Assuntos
Ciclídeos/metabolismo , Monitoramento Ambiental , Poluentes da Água/toxicidade , Animais , Aquicultura , Biomarcadores/metabolismo , Encéfalo/metabolismo , Brasil , Caspase 3/metabolismo , Colinesterases , Glutationa Transferase/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Abrigo para Animais , Fígado/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
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 & purificaçãoRESUMO
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.
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 , Ácido BetulínicoRESUMO
Zinc, at low levels, has several basic housekeeping functions in metalloenzymes, transcription factors, immunoregulation, growth, and cytoprotection, displaying antioxidant, anti-apoptotic, and anti-inflammatory roles. At high levels, however, the metal can be highly toxic. The aim of this work is to investigate the toxic effect of zinc on antioxidant status and stress proteins in the gills of the brown mussel Perna perna exposed for 48 h to zinc chloride (zinc) at 10, 30 and 100 microM. Glutathione reductase (GR) activity was drastically reduced at 30 and 100 microM zinc. At the lower levels, i.e. 10 microM zinc, antioxidant defenses were up-regulated, as were glutathione levels and the activities of glutathione peroxidase and catalase, in spite of the absence of effect on glutathione S-transferase and glucose 6-phosphate dehydrogenase activity. At the higher tested concentration of 100 microM zinc, oxidative stress was apparent as reflected by the increased lipid peroxidation end products and decreased protein thiol and glutathione levels, associated with an inability to up regulate antioxidant defenses. Using 30 microM zinc, higher gill rhodamine B efflux was observed, indicating an activation of multixenobiotic resistance (MXR) activity, which is reinforced by increased immunoreactive P-glycoprotein detection. Zinc also increased the HSP60-immunoreactive protein, whereas the HSP70-immunoreactive protein remained unchanged. Overall, the results indicate that zinc toxicity -- at higher levels -- may be connected to a strong inhibition of GR activity, and related to the pro-oxidative state found. Mussels showed an adaptive-like response to 10 microM zinc by increasing antioxidant defenses. Increased P-glycoprotein and HSP60 expression, and rhodamine B efflux were also remarkable features in the gill response to zinc.
Assuntos
Antioxidantes/metabolismo , Cloretos/toxicidade , Sistema Digestório/efeitos dos fármacos , Brânquias/efeitos dos fármacos , Proteínas de Choque Térmico/metabolismo , Compostos de Zinco/toxicidade , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Animais , Catalase/metabolismo , Chaperonina 60/genética , Chaperonina 60/metabolismo , Sistema Digestório/enzimologia , Sistema Digestório/metabolismo , Brânquias/metabolismo , Glucosefosfato Desidrogenase/metabolismo , Glutationa Transferase/metabolismo , Moluscos , Peroxidase/metabolismo , Rodaminas/metabolismo , Fatores de Tempo , Regulação para CimaRESUMO
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 , EstereoisomerismoRESUMO
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.