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1.
Eur J Med Chem ; 215: 113242, 2021 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-33588180

RESUMO

Currently, SARS-CoV-2 virus is an emerging pathogen that has posed a serious threat to public health worldwide. However, no agents have been approved to treat SARS-CoV-2 infections to date, underscoring the great need for effective and practical therapies for SARS-CoV-2 outbreaks. We reported that a focused screen of OA saponins identified 3-O-ß-chacotriosyl OA benzyl ester 2 as a novel small molecule inhibitor of SARS-CoV-2 virus entry, via binding to SARS-CoV-2 glycoprotein (S). We performed structure-activity relationship profiling of 2 and discovered C-17-COOH of OA was an important modification site that improved both inhibitor potency toward SARS-CoV-2 and selectivity index. Then optimization from hit to lead resulted in a potent fusion inhibitor 12f displaying strong inhibition against infectious SARS-CoV-2 with an IC50 value of 0.97 µM in vitro. Mechanism studies confirmed that inhibition of SARS-CoV-2 viral entry of 12f was mediated by the direct interaction with SARS-CoV-2 S2 subunit to block membrane fusion. These 3-O-ß-chacotriosyl OA amide saponins are suitable for further optimization as SARS-CoV-2 entry inhibitors with the potential to be developed as therapeutic agents for the treatment of SARS-CoV-2 virus infections.


Assuntos
Antivirais/farmacologia , Saponinas/farmacologia , Triterpenos/farmacologia , Internalização do Vírus/efeitos dos fármacos , Animais , Antivirais/síntese química , Antivirais/metabolismo , Chlorocebus aethiops , Descoberta de Drogas , Células HEK293 , Humanos , Testes de Sensibilidade Microbiana , Estrutura Molecular , Ligação Proteica , Subunidades Proteicas/metabolismo , Saponinas/síntese química , Saponinas/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Relação Estrutura-Atividade , Triterpenos/síntese química , Triterpenos/metabolismo , Células Vero
2.
Plant Physiol Biochem ; 160: 166-174, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33497847

RESUMO

Aralia elata (Miq.) Seem is widely used as a medicinal plant and functional food in China. In this study, A. elata plants were exposed to full sunlight (CK), 40% shading (LS), 60% shading (MS), and >80% shading (ES) condition to investigate the effects of shading treatments on growth, stress levels, antioxidant enzymes activity, araloside content and related gene expression. The greatest growth and leaf biomass were achieved in 40% shading, and leaf biomass per plant increased by 16.09% compared to the non-shading treatment. Furthermore, the lowest reactive oxide species (ROS) production and lipid peroxidation resulting from increasing antioxidant enzyme activity were also observed in LS treatment. Overall, shading percentage negatively regulated the expression of key enzymes (squalene synthase, SS; squalene epoxidase, SE and ß-amyrin synthase, bAS) involved in the saponin biosynthesis, resulting in the greatest yields of total and four selected aralosides in A. elata leaves were achieved in sunlight group. However, the greatest yield of total saponin in the leaves was observed in the 40% shading group due to higher leaf biomass. The results suggest that optimizing the field growing conditions would be important for obtaining the greatest yield of bioactive components. Total saponin and selected aralosides also have a significant correlation with ROS production and antioxidant enzyme activity, these indicated the increased yield of these saponins may be part of a defense response. The study concludes that the production of saponin was the interaction of oxidative stress and photosynthesis.


Assuntos
Aralia , Escuridão , Folhas de Planta/efeitos da radiação , Saponinas/metabolismo , Triterpenos/metabolismo , Aralia/genética , Aralia/efeitos da radiação , Regulação da Expressão Gênica de Plantas
3.
Int J Mol Sci ; 21(24)2020 Dec 21.
Artigo em Inglês | MEDLINE | ID: mdl-33371323

RESUMO

The process of fruit ripening involves many chemical changes occurring not only in the mesocarp but also in the epicarp, including changes in the triterpenoid content of fruit cuticular waxes that can modify the susceptibility to pathogens and mechanical properties of the fruit surface. The aim of the study was the determination of the ripening-related changes in the triterpenoid content of fruit cuticular waxes of three plant species from the Rosaceae family, including rugosa rose (Rosa rugosa), black chokeberry (Aronia melanocarpa var. "Galicjanka") and apple (Malus domestica var. "Antonovka"). The triterpenoid and steroid content in chloroform-soluble cuticular waxes was determined by a GC-MS/FID method at four different phenological stages. The profile of identified compounds was rather similar in selected fruit samples with triterpenoids with ursane-, oleanane- and lupane-type carbon skeletons, prevalence of ursolic acid and the composition of steroids. Increasing accumulation of triterpenoids and steroids, as well as the progressive enrichment of the composition of these compounds in cuticular wax during fruit development, was observed. The changes in triterpenoid content resulted from modifications of metabolic pathways, particularly hydroxylation and esterification, that can alter interactions with complementary functional groups of aliphatic constituents and lead to important changes in fruit surface quality.


Assuntos
Frutas/metabolismo , Rosaceae/metabolismo , Triterpenos/metabolismo , Ceras/metabolismo , Frutas/crescimento & desenvolvimento , Redes e Vias Metabólicas , Rosaceae/crescimento & desenvolvimento
4.
Nat Commun ; 11(1): 5664, 2020 11 16.
Artigo em Inglês | MEDLINE | ID: mdl-33199711

RESUMO

Triterpenoid saponins are specialised metabolites distributed widely in the plant kingdom that consist of one or more sugar moieties attached to triterpenoid aglycones. Despite the widely accepted view that glycosylation is catalysed by UDP-dependent glycosyltransferase (UGT), the UGT which catalyses the transfer of the conserved glucuronic acid moiety at the C-3 position of glycyrrhizin and various soyasaponins has not been determined. Here, we report that a cellulose synthase superfamily-derived glycosyltransferase (CSyGT) catalyses 3-O-glucuronosylation of triterpenoid aglycones. Gene co-expression analyses of three legume species (Glycyrrhiza uralensis, Glycine max, and Lotus japonicus) reveal the involvement of CSyGTs in saponin biosynthesis, and we characterise CSyGTs in vivo using Saccharomyces cerevisiae. CSyGT mutants of L. japonicus do not accumulate soyasaponin, but the ectopic expression of endoplasmic reticulum membrane-localised CSyGTs in a L. japonicus mutant background successfully complement soyasaponin biosynthesis. Finally, we produced glycyrrhizin de novo in yeast, paving the way for sustainable production of high-value saponins.


Assuntos
Biocatálise , Glucosiltransferases/metabolismo , Ácido Glucurônico/metabolismo , Saponinas/biossíntese , Vias Biossintéticas , Retículo Endoplasmático/metabolismo , Regulação da Expressão Gênica de Plantas , Glicosilação , Glycyrrhiza uralensis/genética , Ácido Glicirrízico/metabolismo , Funções Verossimilhança , Lotus/genética , Filogenia , Saccharomyces cerevisiae/metabolismo , Saponinas/química , Soja/genética , Especificidade por Substrato , Triterpenos/metabolismo , Uridina Difosfato Ácido Glucurônico/metabolismo
5.
Biomolecules ; 10(11)2020 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-33147723

RESUMO

Plants have been used as drugs to treat human disease for centuries. Ursonic acid (UNA) is a naturally occurring pentacyclic triterpenoid extracted from certain medicinal herbs such as Ziziphus jujuba. Since the pharmacological effects and associated mechanisms of UNA are not well-known, in this work, we attempt to introduce the therapeutic potential of UNA with a comparison to ursolic acid (ULA), a well-known secondary metabolite, for beneficial effects. UNA has a keto group at the C-3 position, which may provide a critical difference for the varied biological activities between UNA and ULA. Several studies previously showed that UNA exerts pharmaceutical effects similar to, or stronger than, ULA, with UNA significantly decreasing the survival and proliferation of various types of cancer cells. UNA has potential to exert inhibitory effects in parasitic protozoa that cause several tropical diseases. UNA also exerts other potential effects, including antihyperglycemic, anti-inflammatory, antiviral, and antioxidant activities. Of note, a recent study highlighted the suppressive potential of UNA against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Molecular modifications of UNA may enhance bioavailability, which is crucial for in vivo and clinical studies. In conclusion, UNA has promising potential to be developed in anticancer and antiprotozoan pharmaceuticals. In-depth investigations may increase the possibility of UNA being developed as a novel reagent for chemotherapy.


Assuntos
Antivirais/farmacologia , Triterpenos/farmacologia , Animais , Anti-Inflamatórios não Esteroides/química , Anti-Inflamatórios não Esteroides/farmacologia , Antineoplásicos Fitogênicos/farmacologia , Antioxidantes/química , Antioxidantes/farmacologia , Antiprotozoários/química , Antiprotozoários/farmacologia , Antivirais/química , Betacoronavirus/efeitos dos fármacos , Betacoronavirus/fisiologia , Humanos , Hipoglicemiantes/química , Hipoglicemiantes/farmacologia , Plantas/química , Triterpenos/química , Triterpenos/metabolismo
6.
Nat Commun ; 11(1): 5354, 2020 10 23.
Artigo em Inglês | MEDLINE | ID: mdl-33097700

RESUMO

Numerous examples of biosynthetic gene clusters (BGCs), including for compounds of agricultural and medicinal importance, have now been discovered in plant genomes. However, little is known about how these complex traits are assembled and diversified. Here, we examine a large number of variants within and between species for a paradigm BGC (the thalianol cluster), which has evolved recently in a common ancestor of the Arabidopsis genus. Comparisons at the species level reveal differences in BGC organization and involvement of auxiliary genes, resulting in production of species-specific triterpenes. Within species, the thalianol cluster is primarily fixed, showing a low frequency of deleterious haplotypes. We further identify chromosomal inversion as a molecular mechanism that may shuffle more distant genes into the cluster, so enabling cluster compaction. Antagonistic natural selection pressures are likely involved in shaping the occurrence and maintenance of this BGC. Our work sheds light on the birth, life and death of complex genetic and metabolic traits in plants.


Assuntos
Arabidopsis/genética , Genoma de Planta , Família Multigênica , Triterpenos/metabolismo , Proteínas de Arabidopsis/genética , Vias Biossintéticas/genética , Evolução Molecular , Genes de Plantas/genética , Metabolismo Secundário
7.
Chem Biol Interact ; 328: 109192, 2020 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-32712081

RESUMO

Many natural products are prodrugs which are biotransformed and activated after oral administration. The investigation of gastrointestinal and hepatic biotransformation can be facilitated by in vitro screening methods. This study compares two widely used in vitro models for hepatic biotransformation: 1) human S9 fractions and 2) human liver microsomes and cytosolic fractions in a two-step sequence, with the purpose of identifying differences in the biotransformation of medicagenic acid, the putative precursor of active metabolites, responsible for the medicinal effects of the herb Herniaria hirsuta. The combination of liquid chromatography coupled to high-resolution mass spectrometry with subsequent suspect and non-target data analysis allowed the identification of thirteen biotransformation products, four of which are reported here for the first time. Eight biotransformation products resulting from oxidative Phase I reactions were identified. Phase II conjugation reactions resulted in the formation of three glucuronidated and two sulfated biotransformation products. No major differences could be observed between incubations with human liver S9 or when utilizing human microsomal and cytosolic fractions. Apart from two metabolites, both methods rendered the same qualitative metabolic profile, with minor quantitative differences. As a result, both protocols applied in this study can be used to study in vitro human liver biotransformation reactions.


Assuntos
Microssomos Hepáticos/metabolismo , Triterpenos/metabolismo , Biotransformação , Avaliação Pré-Clínica de Medicamentos , Feminino , Humanos , Masculino , Frações Subcelulares/metabolismo , Fatores de Tempo , Triterpenos/química
8.
Nat Chem Biol ; 16(7): 783-790, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32393899

RESUMO

Leukotrienes (LT) are lipid mediators of the inflammatory response that are linked to asthma and atherosclerosis. LT biosynthesis is initiated by 5-lipoxygenase (5-LOX) with the assistance of the substrate-binding 5-LOX-activating protein at the nuclear membrane. Here, we contrast the structural and functional consequences of the binding of two natural product inhibitors of 5-LOX. The redox-type inhibitor nordihydroguaiaretic acid (NDGA) is lodged in the 5-LOX active site, now fully exposed by disordering of the helix that caps it in the apo-enzyme. In contrast, the allosteric inhibitor 3-acetyl-11-keto-beta-boswellic acid (AKBA) from frankincense wedges between the membrane-binding and catalytic domains of 5-LOX, some 30 Å from the catalytic iron. While enzyme inhibition by NDGA is robust, AKBA promotes a shift in the regiospecificity, evident in human embryonic kidney 293 cells and in primary immune cells expressing 5-LOX. Our results suggest a new approach to isoform-specific 5-LOX inhibitor development through exploitation of an allosteric site in 5-LOX.


Assuntos
Araquidonato 5-Lipoxigenase/química , Produtos Biológicos/química , Inibidores de Lipoxigenase/química , Masoprocol/química , Triterpenos/química , Sítio Alostérico , Araquidonato 5-Lipoxigenase/genética , Araquidonato 5-Lipoxigenase/metabolismo , Produtos Biológicos/metabolismo , Domínio Catalítico , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Ácidos Hidroxieicosatetraenoicos/química , Ácidos Hidroxieicosatetraenoicos/metabolismo , Leucotrieno B4/química , Leucotrieno B4/metabolismo , Inibidores de Lipoxigenase/metabolismo , Masoprocol/metabolismo , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Triterpenos/metabolismo
9.
J Biosci Bioeng ; 130(2): 142-148, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32327386

RESUMO

As a large group of natural product with significant biological activities, triterpenoid secretion is of particular importance towards its bioproduction. Due to the lack of specific transporters, most triterpenoids are naturally accumulated inside the cells. In this study, by taking an antitumor triterpenoid ganoderic acid 3-hydroxy-lanosta-8,24-dien-26 oic acid (GA-HLDOA) as example, we discovered that addition of 2-hydroxypropyl-ß-cyclodextrin (HP-ß-CD) or 2,6-dimethyl-ß-cyclodextrin (DM-ß-CD) enable the fast and sufficient secretion of GA-HLDOA by the recombinant Saccharomyces cerevisiae strain as constructed in our previous study. In addition, these cyclodextrins (CDs) could not enter into cells, while no significant change of the cell membrane fluidity was observed after CDs treatment. This discovery provides a potential generally applicable method for triterpenoid secretion.


Assuntos
2-Hidroxipropil-beta-Ciclodextrina/farmacologia , Saccharomyces cerevisiae/efeitos dos fármacos , Via Secretória/efeitos dos fármacos , Triterpenos/metabolismo , beta-Ciclodextrinas/farmacologia , Ciclodextrinas/farmacologia , Microbiologia Industrial , Saccharomyces cerevisiae/metabolismo
10.
Plant Mol Biol ; 103(4-5): 489-505, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32306368

RESUMO

KEY MESSAGE: Cucumber plants adapt their transcriptome and metabolome as result of spider mite infestation with opposite consequences for direct and indirect defences in two genotypes. Plants respond to arthropod attack with the rearrangement of their transcriptome which lead to subsequent phenotypic changes in the plants' metabolome. Here, we analysed transcriptomic and metabolite responses of two cucumber (Cucumis sativus) genotypes to chelicerate spider mites (Tetranychus urticae) during the first 3 days of infestation. Genes associated with the metabolism of jasmonates, phenylpropanoids, terpenoids and L-phenylalanine were most strongly upregulated. Also, genes involved in the biosynthesis of precursors for indirect defence-related terpenoids were upregulated while those involved in the biosynthesis of direct defence-related cucurbitacin C were downregulated. Consistent with the observed transcriptional changes, terpenoid emission increased and cucurbitacin C content decreased during early spider-mite herbivory. To further study the regulatory network that underlies induced defence to spider mites, differentially expressed genes that encode transcription factors (TFs) were analysed. Correlation analysis of the expression of TF genes with metabolism-associated genes resulted in putative identification of regulators of herbivore-induced terpenoid, green-leaf volatiles and cucurbitacin biosynthesis. Our data provide a global image of the transcriptional changes in cucumber leaves in response to spider-mite herbivory and that of metabolites that are potentially involved in the regulation of induced direct and indirect defences against spider-mite herbivory.


Assuntos
Cucumis sativus/imunologia , Cucumis sativus/metabolismo , Metaboloma , Infestações por Ácaros/imunologia , Infestações por Ácaros/metabolismo , Tetranychidae , Transcriptoma , Animais , Vias Biossintéticas/genética , Cucumis sativus/genética , Cucumis sativus/parasitologia , Ciclopentanos/metabolismo , Regulação da Expressão Gênica de Plantas , Genes de Plantas/genética , Genoma de Planta , Genótipo , Herbivoria , Oxilipinas/metabolismo , Fenilalanina/metabolismo , Fenilpropionatos/metabolismo , Doenças das Plantas , Folhas de Planta/metabolismo , Metabolismo Secundário/genética , Terpenos/metabolismo , Fatores de Transcrição/genética , Triterpenos/metabolismo , Compostos Orgânicos Voláteis/metabolismo
11.
AAPS PharmSciTech ; 21(4): 123, 2020 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-32337654

RESUMO

The objective of this work was to investigate the capacity of mogroside V (MOG-V), a food additive, as a novel carrier to improve the bioavailability and liver distribution of silybin (SLY). Solid dispersion particles (SDPs) of SLY/MOG-V were prepared utilizing the solvent evaporation method. The physicochemical characterizations of SDPs were evaluated by using dynamic light scattering (DLS), differential scanning calorimetry (DSC), and powder X-ray diffraction (PXRD) measurements. DLS results demonstrated the formation of nanoparticles (206 nm) of SDPs in water. DSC and PXRD analysis revealed that SLY was in amorphous form or molecularly dispersed in SDPs. SDPs also exhibited a major increase in both dissolution rate and saturation solubility, as evidenced by a 1931-fold improvement (2201 µg/mL) in solubility compared with pure SLY (1.14 µg/mL). The pharmacokinetic study in rats showed that oral absorption of SLY/MOG-V SDPs was dramatically increased. The mean value of AUC until 12 h for SLY/MOG-V SDPs (27,481 ng·min/mL) was 24.5-fold higher than that of pure SLY (1122 ng·min/mL). In vivo tissue distribution experiment in mice confirmed that the major distribution tissue was changed from lungs to liver after SLY was loaded into MOG-V. In addition, even orally administrated to mice at a high dose (4.2 g/kg), MOG-V exhibited no undesirable effect on the plasma glucose concentrations. Thus, MOG-V may have the applicability to serve as an ideal excipient for solubilization or as a novel liver targeting carrier for the delivery of SLY.


Assuntos
Portadores de Fármacos/metabolismo , Sistemas de Liberação de Medicamentos/métodos , Fígado/metabolismo , Silibina/metabolismo , Triterpenos/metabolismo , Administração Oral , Animais , Antineoplásicos Fitogênicos/administração & dosagem , Antineoplásicos Fitogênicos/metabolismo , Disponibilidade Biológica , Portadores de Fármacos/administração & dosagem , Avaliação Pré-Clínica de Medicamentos/métodos , Fígado/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos ICR , Ratos , Ratos Sprague-Dawley , Silibina/administração & dosagem , Edulcorantes/administração & dosagem , Edulcorantes/metabolismo , Triterpenos/administração & dosagem , Difração de Raios X/métodos
12.
Plant Sci ; 294: 110433, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32234222

RESUMO

Triterpenoids produced by the secondary metabolism of Betula platyphylla Suk. exhibit important pharmacological activities, such as tumor inhibition, anti-HIV, and defense against pathogens, but the yield of natural synthesis is low, which is insufficient to meet people's needs. In this study, we identified two OSC genes of birch, named as BpCAS and Bpß-AS, respectively. The expression of BpCAS and Bpß-AS were higher levels in roots and in stems, respectively, and they induced expression in response to methyl jasmonate (MeJA), gibberellin (GA3), abscisic acid (ABA), ethylene and mechanical damage. The function of the two genes in the triterpene synthesis of birch was identified by reverse genetics. The inhibition of Bpß-AS gene positively regulates synthesis of betulinic acid. BpCAS interference can significantly promote the upregulation of lupeol synthase gene (BPW) and ß-amyrin synthase gene(BPY), and conversion of 2,3-oxidosqualene to the downstream products betulinic acid and oleanolic acid. This study provided a basis for the genetic improvement of triterpenoid synthesis in birch through genetic engineering. The obtained transgenic birch and suspension cells served as material resources for birch triterpenoid applications in further.


Assuntos
Betula/metabolismo , Triterpenos/metabolismo , Ácido Abscísico/farmacologia , Acetatos/farmacologia , Betula/efeitos dos fármacos , Ciclopentanos/farmacologia , Regulação da Expressão Gênica de Plantas , Giberelinas/farmacologia , Transferases Intramoleculares/genética , Transferases Intramoleculares/metabolismo , Ácido Oleanólico/metabolismo , Oxilipinas/farmacologia , Triterpenos Pentacíclicos , Esqualeno/análogos & derivados , Esqualeno/metabolismo
13.
Planta ; 251(4): 78, 2020 Mar 10.
Artigo em Inglês | MEDLINE | ID: mdl-32157441

RESUMO

MAIN CONCLUSION: A hypothesis that squalene cyclase genes are widely distributed throughout ferns was proposed. We successfully isolated a squalene cyclase pseudogene from a fern from which no triterpene hydrocarbons were detected Ferns are the most primitive vascular plants, with their locations ranging from tropical to cold temperate regions and from lowland to alpine zones. The triterpene hydrocarbons and their derivatives are characteristic fern metabolites, and are also chemophenetic markers. Recently, our biosynthetic study into fern squalene cyclases (SCs), the enzymes responsible for triterpene synthesis, gave an unexpected inconsistency between genotype (enzyme function) and chemotype (triterpene profile). This finding prompted us to propose a hypothesis that SC genes are widely distributed throughout ferns and lycophytes whether or not they produce triterpene hydrocarbons. To test this hypothesis, we employed a multifaceted approach based on phytochemical, biochemical, and phylogenetic analyses. As anticipated, we successfully isolated two SC pseudogenes from a fern from in which no or only one triterpene hydrocarbon was detected. Subsequent mutagenesis experiments resulted in the functional conversion of these pseudogenes into active SC genes. Given an auxiliary hypothesis regarding the inherent limit of the degenerate polymerase chain reaction (PCR) method, the overall dataset supported our hypothesis, although correction was required with respect to plant coverage. Not only did the corrected hypothesis outline the distribution of SC genes throughout ferns, it provided insight into the molecular basis of the triterpene-based chemophenetics in ferns, which is also discussed.


Assuntos
Gleiquênias/metabolismo , Triterpenos/metabolismo , Clonagem Molecular , Evolução Molecular , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Liases/genética , Liases/metabolismo , Espectroscopia de Ressonância Magnética , Biologia Molecular , Filogenia , Pichia/genética , Alinhamento de Sequência
14.
Sci Rep ; 10(1): 4204, 2020 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-32144288

RESUMO

Diatoms are ubiquitous microalgae that have developed remarkable metabolic plasticity and gene diversification. Here we report the first elucidation of the complete biosynthesis of sterols in the lineage. The study has been carried out on the bloom-forming species Skeletonema marinoi and Cyclotella cryptica that synthesise an ensemble of sterols with chemotypes of animals (cholesterol and desmosterol), plants (dihydrobrassicasterol and 24-methylene cholesterol), algae (fucosterol) and marine invertebrates (clionasterol). In both species, sterols derive from mevalonate through cyclization of squalene to cycloartenol by cycloartenol synthase. The pathway anticipates synthesis of cholesterol by enzymes of the phytosterol route in plants, as recently reported in Solanaceae. Major divergences stem from reduction of Δ24(28) and Δ24(25) double bonds which, in diatoms, are apparently dependent on sterol reductases of fungi, algae and animals. Phylogenetic comparison revealed a good level of similarity between the sterol biosynthetic genes of S. marinoi and C. cryptica with those in the genomes of the other diatoms sequenced so far.


Assuntos
Diatomáceas/metabolismo , Esteróis/metabolismo , Animais , Transferases Intramoleculares/metabolismo , Ácido Mevalônico/metabolismo , Filogenia , Fitosteróis/metabolismo , Transdução de Sinais/fisiologia , Solanaceae/metabolismo , Esqualeno/metabolismo , Triterpenos/metabolismo
15.
Arch Biochem Biophys ; 684: 108314, 2020 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-32088220

RESUMO

Signal transducer and activator of transcription 3 (STAT3) is expressed aberrantly in multiple tumors, including gastric cancer (GC). STAT3 overexpression and excessive activation have been confirmed to play vital roles in tumorigenesis. Cucurbitacin B (CuB) is a natural product with potent anti-cancer activities in solid tumors. Here, we systematically studied the underlying molecular mechanisms of CuB inhibition of GC both in vitro and in vivo. In GC cell lines, nanomolar concentrations of CuB decreased the phosphorylation of TYR-705 in STAT3 and suppressed STAT3 target gene expression, including c-Myc and Bcl-xL. Computational docking analysis showed that CuB interacts with the DNA-binding domain of STAT3 at several hydrophobic residues. In addition, pull-down experiments showed that CuB is a direct inhibitor of STAT3. CuB in combination with the conventional chemotherapy drug cisplatin exerted enhanced cytotoxicity in GC cells, possibly due to the potentiated inhibition of STAT3 activation. Moreover, a xenograft mouse model confirmed the therapeutic effect of CuB in vivo. These characteristics render CuB a promising candidate drug for further development in the design of new effective STAT3 inhibitors for treating GC.


Assuntos
Antineoplásicos/uso terapêutico , Fator de Transcrição STAT3/antagonistas & inibidores , Neoplasias Gástricas/tratamento farmacológico , Triterpenos/uso terapêutico , Animais , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Cisplatino/farmacologia , Feminino , Humanos , Masculino , Camundongos Nus , Simulação de Acoplamento Molecular , Invasividade Neoplásica , Ligação Proteica , Fator de Transcrição STAT3/metabolismo , Neoplasias Gástricas/patologia , Triterpenos/metabolismo , Triterpenos/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto
16.
Int J Mol Sci ; 21(4)2020 Feb 11.
Artigo em Inglês | MEDLINE | ID: mdl-32054089

RESUMO

Platycosides, the saponins abundant in Platycodi radix (the root of Platycodon grandiflorum), have diverse pharmacological activities and have been used as food supplements. Since deglycosylated saponins exhibit higher biological activity than glycosylated saponins, efforts are on to enzymatically convert glycosylated platycosides to deglycosylated platycosides; however, the lack of diversity and specificities of these enzymes has limited the kinds of platycosides that can be deglycosylated. In the present study, we examined the enzymatic conversion of platycosides and showed that Cytolase PCL5 completely converted platycoside E and polygalacin D3 into deapiose-xylosylated platycodin D and deapiose-xylosylated polygalacin D, respectively, which were identified by LC-MS analysis. The platycoside substrates were hydrolyzed through the following novel hydrolytic pathways: platycoside E → platycodin D3 → platycodin D → deapiosylated platycodin D → deapiose-xylosylated platycodin D; and polygalacin D3 → polygalacin D → deapiosylated polygalacin D → deapiose-xylosylated polygalacin D. Our results show that cytolast PCL5 may have a potential role in the development of biologically active platycosides that may be used for their diverse pharmacological activities.


Assuntos
Ácido Oleanólico/análogos & derivados , Proteínas de Plantas/metabolismo , Platycodon/metabolismo , Saponinas/metabolismo , Triterpenos/metabolismo , Glicosilação , Ácido Oleanólico/metabolismo , Platycodon/enzimologia
17.
Nat Commun ; 11(1): 868, 2020 02 13.
Artigo em Inglês | MEDLINE | ID: mdl-32054834

RESUMO

Synthetic biology, genome engineering and directed evolution offer innumerable tools to expedite engineering of strains for optimising biosynthetic pathways. One of the most radical is SCRaMbLE, a system of inducible in vivo deletion and rearrangement of synthetic yeast chromosomes, diversifying the genotype of millions of Saccharomyces cerevisiae cells in hours. SCRaMbLE can yield strains with improved biosynthetic phenotypes but is limited by screening capabilities. To address this bottleneck, we combine automated sample preparation, an ultra-fast 84-second LC-MS method, and barcoded nanopore sequencing to rapidly isolate and characterise the best performing strains. Here, we use SCRaMbLE to optimise yeast strains engineered to produce the triterpenoid betulinic acid. Our semi-automated workflow screens 1,000 colonies, identifying and sequencing 12 strains with between 2- to 7-fold improvement in betulinic acid titre. The broad applicability of this workflow to rapidly isolate improved strains from a variant library makes this a valuable tool for biotechnology.


Assuntos
Genes Sintéticos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Triterpenos/metabolismo , Biotecnologia , Cromatografia Líquida , Cromossomos Fúngicos , Evolução Molecular Direcionada , Biblioteca Gênica , Estudos de Associação Genética , Engenharia Genética , Testes Genéticos , Genoma Fúngico , Espectrometria de Massas , Triterpenos Pentacíclicos , Recombinação Genética , Biologia Sintética
18.
Nutrients ; 12(2)2020 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-32013132

RESUMO

A wide range of people in the world use natural remedies as primary approaches against illnesses. Accordingly, understanding the mechanisms of action of phytochemicals has become of great interest. In this context, Centella asiatica L. is extensively used, not only as anti-inflammatory or antioxidant agent but also as brain tonic. On this basis, the purpose of this study was to evaluate whether the chronic administration of C. asiatica L. to adult male rats was able to improve the expression of Bdnf, one of the main mediators of brain plasticity. Moreover, we assessed whether the treatment could affect the cognitive performance in the novel object recognition (NOR) test. We confirmed the presence of the main compounds in the plasma. Furthermore, C. asiatica L. administration induced an increase of Bdnf in the prefrontal cortex, and the administration of the higher dose of the extract was able to improve cognitive performance. Finally, the increase in the preference index in the NOR test was paralleled by a further increase in Bdnf expression. Overall, we highlight the ability of C. asiatica L. to affect brain functions by increasing Bdnf expression and by enhancing the cognitive performance.


Assuntos
Fator Neurotrófico Derivado do Encéfalo/metabolismo , Centella/química , Cognição/efeitos dos fármacos , Córtex Pré-Frontal/efeitos dos fármacos , Triterpenos/farmacologia , Animais , Antígenos de Diferenciação/genética , Antígenos de Diferenciação/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Fator Neurotrófico Derivado do Encéfalo/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Masculino , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Triterpenos/sangue , Triterpenos/metabolismo
19.
Mol Biotechnol ; 62(2): 132-141, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31897972

RESUMO

A farnesyl diphosphate synthase (FPS) cDNA and promoter region was cloned from Sanghuangporus baumii. The gene contains a 150-bp 5'-untranslated region (UTR), a 154-bp 3'-UTR, and a 1062-bp open reading frame (ORF) encoding a 354 amino acid polypeptide. The FPS-DNA includes three exons (nucleotides 1 -123, 184-321, and 505-1305) and two introns (nucleotides 124-183 and 322-504). The FPS protein has a molecular weight of 40.73 kDa, it is hydrophilic with a theoretical isoelectric point of 5.13, and the secondary and three-dimensional structure were analysed. There is a transcription start site at nucleotides 1318-1368 of the promoter, which includes typical eukaryotic promoter elements (TATA Box, CAAT Box, ARBE, AT-rich element, G-box, MBS, Sp1, LTR). FPS was expressed in Escherichia coli BL21, and the recombinant protein (63.41 kDa) was subjected to dodecyl sulphate, sodium salt-polyacrylamide gel electrophoresis (SDS-PAGE). FPS transcription was measured during different developmental stages, and expression in 11 and 13 days mycelia was upregulated 49.3-fold and 125.4-fold, respectively, compared with 9 days mycelia controls. Through analysing, S. baumii triterpenoid content was correlated with the transcription level of FPS during different development stages, and the triterpenoid content peaked at day 15 (7.21 mg/g).


Assuntos
Basidiomycota/enzimologia , Geraniltranstransferase/metabolismo , Triterpenos/metabolismo , Regiões 3' não Traduzidas , Regiões 5' não Traduzidas , Sequência de Aminoácidos/genética , Basidiomycota/genética , Basidiomycota/crescimento & desenvolvimento , Clonagem Molecular , Escherichia coli , Éxons , Expressão Gênica , Geraniltranstransferase/química , Geraniltranstransferase/genética , Íntrons , Filogenia , Regiões Promotoras Genéticas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Triterpenos/farmacologia
20.
Microb Cell Fact ; 19(1): 15, 2020 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-31992268

RESUMO

BACKGROUND: Celastrol is a promising anti-obesity agent that acts as a sensitizer of the protein hormone leptin. Despite its potent activity, a sustainable source of celastrol and celastrol derivatives for further pharmacological studies is lacking. RESULTS: To elucidate the celastrol biosynthetic pathway and reconstruct it in Saccharomyces cerevisiae, we mined a root-transcriptome of Tripterygium wilfordii and identified four oxidosqualene cyclases and 49 cytochrome P450s as candidates to be involved in the early steps of celastrol biosynthesis. Using functional screening of the candidate genes in Nicotiana benthamiana, TwOSC4 was characterized as a novel oxidosqualene cyclase that produces friedelin, the presumed triterpenoid backbone of celastrol. In addition, three P450s (CYP712K1, CYP712K2, and CYP712K3) that act downstream of TwOSC4 were found to effectively oxidize friedelin and form the likely celastrol biosynthesis intermediates 29-hydroxy-friedelin and polpunonic acid. To facilitate production of friedelin, the yeast strain AM254 was constructed by deleting UBC7, which afforded a fivefold increase in friedelin titer. This platform was further expanded with CYP712K1 to produce polpunonic acid and a method for the facile extraction of products from the yeast culture medium, resulting in polpunonic acid titers of 1.4 mg/L. CONCLUSION: Our study elucidates the early steps of celastrol biosynthesis and paves the way for future biotechnological production of this pharmacologically promising compound in engineered yeast strains.


Assuntos
Fármacos Antiobesidade/metabolismo , Biotecnologia/métodos , Tabaco/metabolismo , Tripterygium/metabolismo , Triterpenos/metabolismo , Clonagem Molecular , Sistema Enzimático do Citocromo P-450/metabolismo , Saccharomyces cerevisiae/genética , Terpenos/metabolismo
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