RESUMO
Pseudomonas protegens can generally produce multiple antibiotics including pyoluteorin (Plt), 2,4-diacetylphloroglucinol (DAPG), and pyrrolnitrin (Prn). In this study, we discovered and characterized a quorum sensing (QS) system, PpqI/R, in P. protegens H78. PpqI/R, encoded by two open reading frames (ORFs) (H78_01960/01961) in P. protegens H78 genome, is a LuxI/R-type QS system. Four long-chain acyl homoserine lactone (AHL) signaling molecules, 3-OH-C10-HSL, 3-OH-C12-HSL, C12-HSL, and 3-OH-C14-HSL, are produced by H78. Biosynthesis of these AHLs is catalyzed by PpqI synthase and activated by the PpqR regulator in H78 and in Escherichia coli when heterologously expressed. PpqR activates ppqI expression by targeting the lux box upstream of the ppqI promoter in cooperation with corresponding AHLs. The four aforementioned AHLs exhibited different capabilities to induce ppqI promoter expression, with 3-OH-C12-HSL showing the highest induction activity. In H78 cells, ppqI/R expression is activated by the two-component system GacS/A and the RNA chaperone Hfq. Differential regulation of the PpqI/R system in secondary metabolism has a negative effect on DAPG biosynthesis and ped operon (involved in volatile organic compound biosynthesis) expression. In contrast, Plt biosynthesis and prn operon expression were positively regulated by PpqI/R. In summary, PpqI/R, the first characterized QS system in P. protegens, is activated by GacS/A and Hfq and controls the expression of secondary metabolites, including antibiotics.
Assuntos
Proteínas de Bactérias , Regulação Bacteriana da Expressão Gênica , Pseudomonas , Percepção de Quorum , Percepção de Quorum/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Pseudomonas/metabolismo , Pseudomonas/genética , Fator Proteico 1 do Hospedeiro/metabolismo , Fator Proteico 1 do Hospedeiro/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Floroglucinol/metabolismo , Floroglucinol/análogos & derivados , Acil-Butirolactonas/metabolismo , Fenóis/metabolismo , Pirrolnitrina/metabolismo , Pirróis/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Fases de Leitura Aberta , Regiões Promotoras Genéticas , Compostos Heterocíclicos com 3 Anéis/metabolismoRESUMO
Phytochemical study on the flowers of Hypericum formosanum Maxim. (Hypericaceae) led to the isolation of formohyperins G-L (1-6), whose structures were assigned by detailed spectroscopic analysis. Formohyperins G-L (1-6) are new benzoylphloroglucinols substituted by a C10 unit, a prenyl group, and a methyl group. Formohyperins G-J (1-4) possess a 6/6/6-tricyclic structure, while formohyperins K (5) and L (6) have a unique 6/6/5/4-tetracyclic structure consisting of cyclohexadienone, dihydropyrane, cyclopentane, and cyclobutane rings. The absolute configurations of 1-6 were deduced by analysis of the ECD spectra. Formohyperins G-J (1-4) and L (6) were found to show potent inhibitory activities against IL-1ß release from LPS-treated murine microglial cells with EC50 values of 5.0, 10.9, 6.3, 10.8, and 13.7 µM, respectively, without cytotoxicity. 6-O-Methylformohyperins G (1a) and I (3a) also exhibited the inhibitory activities with EC50 values of 4.7 and 2.7 µM, respectively, although they were cytotoxic against microglial cells.
Assuntos
Flores , Hypericum , Floroglucinol , Hypericum/química , Animais , Floroglucinol/química , Floroglucinol/farmacologia , Flores/química , Camundongos , Estrutura Molecular , Interleucina-1beta/metabolismo , Microglia/efeitos dos fármacos , Microglia/metabolismo , Prenilação , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Lipopolissacarídeos/farmacologia , Linhagem CelularRESUMO
Advanced glycation end products (AGEs) play an important role in the pathogenesis of age-linked disorders and diabetes mellitus. The aim of this study was to assess the repurposing potential of Phloroglucinol (PHL the antispasmodic drug), as an anti-glycation agent using Fructose-BSA model. The ability of PHL to inhibit AGE formation was evaluated using AGEs formation (Intrinsic fluorescence), fructosamine adduct (NBT) and free lysine availability (TNBSA) assays. The BSA protein conformation was assessed through Thioflavin-T, Congo-Red and Circular Dichroism assays. The lysine blockade and carbonyl entrapment were explored as possible mode of action. Our data showed that PHL significantly decreased the formation of AGEs with an IC50 value of 0.3mM. The fructosamine adducts and free lysine load was found to be reduced. Additionally, the BSA conformation was preserved by PHL. Mechanistic assays did not reveal involvement of lysine blockade as underlying reason for reduction in AGEs load. This was also supported by computational data whereby PHL failed to engage any catalytic residue involved in early fructose-BSA interaction. However, it was found to entrap the carbonyl moieties. In conclusion, the PHL demonstrated anti-glycation potential, which can be attributed to its ability to entrap carbonyl intermediates. Hence, the clinically available antispasmodic drug, presents itself as a promising candidate to be repurposed as anti-glycation agent.
Assuntos
Produtos Finais de Glicação Avançada , Floroglucinol , Soroalbumina Bovina , Produtos Finais de Glicação Avançada/metabolismo , Soroalbumina Bovina/química , Soroalbumina Bovina/metabolismo , Floroglucinol/farmacologia , Floroglucinol/química , Glicosilação/efeitos dos fármacos , Lisina/metabolismo , Lisina/química , Frutose/química , Frutose/metabolismo , Animais , Frutosamina/metabolismo , Simulação de Acoplamento Molecular , BovinosRESUMO
Phloroglucinol (1,3,5-trihydroxybenzene) is a key intermediate in the degradation of polyphenols such as flavonoids and hydrolysable tannins and can be used by certain bacteria as a carbon and energy source for growth. The identification of enzymes that participate in the fermentation of phloroglucinol to acetate and butyrate in Clostridia was recently reported. In this study, we present the discovery and characterization of a novel metabolic pathway for phloroglucinol degradation in the bacterium Collinsella sp. zg1085, from marmot respiratory tract. In both the Clostridial and Collinsella pathways, phloroglucinol is first reduced to dihydrophoroglucinol by the NADPH-dependent phloroglucinol reductase (PGR), followed by ring opening to form (S)-3-hydroxy-5-oxohexanoate by a Mn2+-dependent dihydrophloroglucinol cyclohydrolase (DPGC). In the Collinsella pathway, (S)-3-hydroxy-5-oxohexanoate is then cleaved to form malonate semialdehyde and acetone by a newly identified aldolase (HOHA). Finally, a NADP+-dependent malonate-semialdehyde dehydrogenase converts malonate semialdehyde to CO2 and acetyl-CoA, an intermediate in carbon and energy metabolism. Recombinant expression of the Collinsella PGR, DPGC, and HOHA in E. coli enabled the conversion of phloroglucinol into acetone, providing support for the proposed pathway. Experiments with Olsenella profusa, another bacterium containing the gene cluster of interest, show that the PGR, DPGC, HOHA, and MSDH are induced by phloroglucinol. Our findings add to the variety of metabolic pathways for the degradation of phloroglucinol, a widely distributed phenolic compound, in the anaerobic microbiome.IMPORTANCEPhloroglucinol is an important intermediate in the bacterial degradation of polyphenols, a highly abundant class of plant natural products. Recent research has identified key enzymes of the phloroglucinol degradation pathway in butyrate-producing anaerobic bacteria, which involves cleavage of a linear triketide intermediate by a beta ketoacid cleavage enzyme, requiring acetyl-CoA as a co-substrate. This paper reports a variant of the pathway in the lactic acid bacterium Collinsella sp. zg1085, which involves cleavage of the triketide intermediate by a homolog of deoxyribose-5-phosphate aldolase, highlighting the variety of mechanisms for phloroglucinol degradation by different anaerobic bacterial taxa.
Assuntos
Redes e Vias Metabólicas , Floroglucinol , Floroglucinol/metabolismo , Floroglucinol/análogos & derivados , Redes e Vias Metabólicas/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Aldeído Liases/metabolismo , Aldeído Liases/genética , AnimaisRESUMO
Six new polycyclic polyprenylated acylphloroglucinols (PPAPs), hyperidiones A-F (1-6), were obtained from Hypericum perforatum L. Their structures were characterized via extensive spectroscopic analyses, the circular dichroism data of the in situ formed [Mo2(OCOCH3)4] complexes, the nuclear magnetic resonance calculation with DP4 + probability analysis, and the calculated electronic circular dichroism (ECD) spectra. Compounds 1-6 are bicyclic polyprenylated acylphloroglucinols with a major bicyclo[3.3.1]nonane-2,4,9-trione skeleton. Notably, compound 1 is a rare PPAP with a hydroperoxy group, and a plausible biosynthetic pathway for 1 was proposed. Compounds 4 and 6 exhibited significant neuroprotective effects under 10 µM against corticosterone (CORT)-injured SH-SY5Y cells. Furthermore, compound 4 demonstrated a noteworthy antidepressant effect at the dose of 5 mg/kg in the tail suspension test (TST) of mice, which was equivalent to 5 mg/kg of fluoxetine. And it potentially exerted an antidepressant effect through the hypothalamic-pituitary-adrenal (HPA) axis.
Assuntos
Antidepressivos , Hypericum , Floroglucinol , Hypericum/química , Antidepressivos/farmacologia , Antidepressivos/química , Antidepressivos/isolamento & purificação , Animais , Floroglucinol/farmacologia , Floroglucinol/química , Floroglucinol/isolamento & purificação , Camundongos , Humanos , Estrutura Molecular , Fármacos Neuroprotetores/farmacologia , Fármacos Neuroprotetores/química , Fármacos Neuroprotetores/isolamento & purificação , Relação Estrutura-Atividade , Relação Dose-Resposta a Droga , Masculino , Linhagem Celular Tumoral , Compostos Policíclicos/farmacologia , Compostos Policíclicos/química , Compostos Policíclicos/isolamento & purificação , Corticosterona , Elevação dos Membros PosterioresRESUMO
Nanoplastics (NPs), plastic particles ranging from 1 to 100 nm are ubiquitous environmental pollutants infiltrating ecosystems. Their small size and widespread use in various products raise concerns for human health, particularly their association with cardiovascular diseases (CVD). NPs can enter the human body through multiple routes, causing oxidative stress, and leading to the senescence and dysfunction of endothelial cells (ECs). Although there are potential natural compounds for treating CVD, there is limited research on preventing CVD induced by NPs. This study investigates the efficacy of Ecklonia cava extract (ECE) in preventing NPs-induced premature vascular senescence and dysfunction. Exposure of porcine coronary arteries (PCAs) and porcine coronary ECs to NPs, either alone or in combination with ECE, demonstrated that ECE mitigates senescence-associated ß-galactosidase (SA-ß-gal) activity induced by NPs, thus preventing premature endothelial senescence. ECE also improved NPs-induced vascular dysfunction. The identified active ingredient in Ecklonia cava, 2,7'-Phloroglucinol-6,6'-bieckol (PHB), a phlorotannin, proved to be pivotal in these protective effects. PHB treatment ameliorated SA-ß-gal activity, reduced oxidative stress, restored cell proliferation, and decreased the expression of cell cycle regulatory proteins such as p53, p21, p16, and angiotensin type 1 receptor (AT1), well known triggers for EC senescence. Moreover, PHB also improved NPs-induced vascular dysfunction by upregulating endothelial nitric oxide synthase (eNOS) expression and restoring endothelium-dependent vasorelaxation. In conclusion, Ecklonia cava and its active ingredient, PHB, exhibit potential as therapeutic agents against NPs-induced premature EC senescence and dysfunction, indicating a protective effect against environmental pollutants-induced CVDs associated with vascular dysfunction.
Assuntos
Senescência Celular , Dioxinas , Phaeophyceae , Senescência Celular/efeitos dos fármacos , Animais , Suínos , Células Endoteliais/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Floroglucinol/farmacologia , Floroglucinol/análogos & derivados , NanopartículasRESUMO
The role of antagonistic secondary metabolites produced by Pseudomonas protegens in suppression of soil-borne phytopathogens has been clearly documented. However, their contribution to the ability of P. protegens to establish in soil and rhizosphere microbiomes remains less clear. Here, we use a four-species synthetic community (SynCom) in which individual members are sensitive towards key P. protegens antimicrobial metabolites (DAPG, pyoluteorin, and orfamide A) to determine how antibiotic production contributes to P. protegens community invasion and to identify community traits that counteract the antimicrobial effects. We show that P. protegens readily invades and alters the SynCom composition over time, and that P. protegens establishment requires production of DAPG and pyoluteorin. An orfamide A-deficient mutant of P. protegens invades the community as efficiently as wildtype, and both cause similar perturbations to community composition. Here, we identify the microbial interactions underlying the absence of an orfamide A mediated impact on the otherwise antibiotic-sensitive SynCom member, and show that the cyclic lipopeptide is inactivated and degraded by the combined action of Rhodococcus globerulus D757 and Stenotrophomonas indicatrix D763. Altogether, the demonstration that the synthetic community constrains P. protegens invasion by detoxifying its antibiotics may provide a mechanistic explanation to inconsistencies in biocontrol effectiveness in situ.
Assuntos
Biotransformação , Pseudomonas , Metabolismo Secundário , Microbiologia do Solo , Pseudomonas/metabolismo , Pseudomonas/genética , Rizosfera , Microbiota , Interações Microbianas , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Fenóis , Floroglucinol/análogos & derivados , PirróisRESUMO
OBJECTIVE: The continuously increasing extracellular matrix stiffness during intervertebral disc degeneration promotes disease progression. In an attempt to obtain novel treatment methods, this study aims to investigate the changes in nucleus pulposus cells under the stimulation of a stiff microenvironment. DESIGN: RNA sequencing and metabolomics experiments were combined to evaluate the primary nucleus pulposus and screen key targets under mechanical biological stimulation. Additionally, small molecules work in vitro were used to confirm the target regulatory effect and investigate the mechanism. In vivo, treatment effects were validated using a rat caudal vertebrae compression model. RESULTS: Our research results revealed that by activating TRPC6, hyperforin, a herbaceous extract can rescue the inflammatory phenotype caused by the stiff microenvironment, hence reducing intervertebral disc degeneration (IDD). Mechanically, it activates mitochondrial fission to inhibit PFKFB3. CONCLUSION: In summary, this study reveals the important bridging role of TRPC6 between mechanical stiffness, metabolism, and inflammation in the context of nucleus pulposus degeneration. TRPC6 activation with hyperforin may become a promising treatment for IDD.
Assuntos
Matriz Extracelular , Degeneração do Disco Intervertebral , Dinâmica Mitocondrial , Núcleo Pulposo , Floroglucinol , Ratos Sprague-Dawley , Animais , Núcleo Pulposo/metabolismo , Núcleo Pulposo/patologia , Núcleo Pulposo/efeitos dos fármacos , Degeneração do Disco Intervertebral/tratamento farmacológico , Degeneração do Disco Intervertebral/patologia , Degeneração do Disco Intervertebral/metabolismo , Ratos , Floroglucinol/farmacologia , Floroglucinol/análogos & derivados , Floroglucinol/uso terapêutico , Dinâmica Mitocondrial/efeitos dos fármacos , Matriz Extracelular/metabolismo , Matriz Extracelular/efeitos dos fármacos , Masculino , Células Cultivadas , Humanos , Terpenos/farmacologia , Terpenos/uso terapêutico , Canais de Cátion TRPC/metabolismo , Modelos Animais de Doenças , Inflamação/tratamento farmacológicoRESUMO
In response to the pressing global challenge of antibiotic resistance, time efficient design and synthesis of novel antibiotics are of immense need. Polycyclic polyprenylated acylphloroglucinols (PPAP) were previously reported to effectively combat a range of gram-positive bacteria. Although the exact mode of action is still not clear, we conceptualized a late-stage divergent synthesis approach to expand our natural product-based PPAP library by 30 additional entities to perform SAR studies against methicillin-resistant Staphylococcus aureus (MRSA). Although at this point only data from cellular assays are available and understanding of molecular drug-target interactions are lacking, the experimental data were used to generate 3D-QSAR models via an artificial intelligence training and to identify a common pharmacophore model. The experimentally validated QSAR model enabled the estimation of anti-MRSA activities of a virtual compound library consisting of more than 100,000 in-silico generated B PPAPs, out of which the 20 most promising candidates were synthesized. These novel PPAPs revealed significantly improved cellular activities against MRSA with growth inhibition down to concentrations less than 1â µm.
Assuntos
Antibacterianos , Produtos Biológicos , Staphylococcus aureus Resistente à Meticilina , Testes de Sensibilidade Microbiana , Floroglucinol , Relação Quantitativa Estrutura-Atividade , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Antibacterianos/farmacologia , Antibacterianos/química , Antibacterianos/síntese química , Produtos Biológicos/química , Produtos Biológicos/farmacologia , Produtos Biológicos/síntese química , Floroglucinol/química , Floroglucinol/farmacologia , Floroglucinol/síntese química , Desenho de Fármacos , Compostos Policíclicos/química , Compostos Policíclicos/farmacologia , Compostos Policíclicos/síntese químicaRESUMO
Many polyprenylated acylphloroglucinols with fascinating chemical structures and intriguing biological activities have been identified as key to phytochemicals isolated from Garcinia, Hypericum, and related genera. In the present work, two chiral, tautomeric, highly-oxygenated polyprenylated acylphloroglucinols tethered with acyl and prenyl moieties on a bicyclo[3.3.1]nonanetrione core were isolated from the 95% ethanolic extract of Garcinia gummi-gutta fruit. The structures of both compounds were elucidated based on the NMR and MS data with ambiguity in the exact position of the enol and keto functions at C-1 and C-3 of the core structure. The structures of both polyprenylated acylphloroglucinols were established as a structurally revised guttiferone J and the new iso-guttiferone J with the aid of gauge-independent atomic orbital NMR calculations, CP3 probability analyses, specific rotation calculations, and electronic circular dichroism calculations in combination with the experimental data. The structures of both compounds resemble hyperforin, a potent activator of the human pregnane X receptor. As expected, both compounds showed strong pregnane X receptor activation at 10 µM [7.1-fold (guttiferone J) and 5.0-fold (iso-guttiferone J)], explained by a molecular docking study, necessitating further in-depth investigation to substantiate the herb-drug interaction potential of G. gummi-gutta upon co-administration with pharmaceutical drugs.
Assuntos
Garcinia , Espectroscopia de Ressonância Magnética , Garcinia/química , Estrutura Molecular , Frutas/química , Benzofenonas/química , Benzofenonas/isolamento & purificação , Benzofenonas/farmacologia , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Compostos Fitoquímicos/isolamento & purificação , Compostos Fitoquímicos/química , Compostos Fitoquímicos/farmacologia , Floroglucinol/química , Floroglucinol/isolamento & purificação , HumanosRESUMO
AIM: Biotechnical processes in Escherichia coli often operate with artificial plasmids. However, these bioprocesses frequently encounter plasmid loss. To ensure stable expression of heterologous genes in E. coli BL21(DE3), a novel plasmid addiction system (PAS) was developed. METHODS AND RESULTS: This PAS employed an essential gene grpE encoding a cochaperone in the DnaK-DnaJ-GrpE chaperone system as the selection marker, which represented a chromosomal ΔgrpE mutant harboring episomal expression plasmids that carry supplementary grpE alleles to restore the deficiency. To demonstrate the feasibility of this system, it was implemented in phloroglucinol (PG) biosynthesis, manifesting improved host tolerance to PG and increased PG production. Specifically, PG titer significantly improved from 0.78 ± 0.02 to 1.34 ± 0.04 g l-1, representing a 71.8% increase in shake-flask fermentation. In fed-batch fermentation, the titer increased from 3.71 ± 0.11 to 4.54 ± 0.10 g l-1, showing a 22.4% increase. RNA sequencing and transcriptome analysis revealed that the improvements were attributed to grpE overexpression and upregulation of various protective chaperones and the biotin acetyl-CoA carboxylase ligase coding gene birA. CONCLUSION: This novel PAS could be regarded as a typical example of nonanabolite- and nonmetabolite-related PAS. It effectively promoted plasmid maintenance in the host, improved tolerance to PG, and increased the titer of this compound.
Assuntos
Proteínas de Escherichia coli , Proteínas de Choque Térmico , Floroglucinol , Plasmídeos , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Fermentação , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Floroglucinol/metabolismo , Floroglucinol/análogos & derivados , Plasmídeos/genéticaRESUMO
Seven pairs of undescribed monoterpenoid polyprenylated acylphloroglucinol enantiomers [(±)-hypermonanones A-G (1-7)], together with three known analogues, were identified from the whole plant of Hypericum monanthemum Hook. The structures of these compounds were determined by analyses of their UV, HRESIMS, 1D/2D NMR spectroscopic data, and NMR calculations. The absolute configurations of these compounds were assigned by ECD calculations after chiral HPLC separation. Diverse monoterpene moieties were fused at C-3/C-4 of the dearomatized acylphloroglucinol core, which led to 3,4-dihydro-2H-pyran-integrated angular or linear type 6/6/6 tricyclic skeletons in 1-7. Compounds (-)-2 and (+)-2 exhibited significant NO inhibitory activity against LPS induced RAW264.7 cells with the IC50 values of 7.07 ± 1.02 µM and 11.39 ± 0.24 µM, respectively.
Assuntos
Hypericum , Monoterpenos , Floroglucinol , Compostos Fitoquímicos , Hypericum/química , Camundongos , Estrutura Molecular , Monoterpenos/isolamento & purificação , Monoterpenos/farmacologia , Floroglucinol/isolamento & purificação , Floroglucinol/farmacologia , Floroglucinol/química , Células RAW 264.7 , Compostos Fitoquímicos/farmacologia , Compostos Fitoquímicos/isolamento & purificação , Animais , Óxido Nítrico/metabolismo , Estereoisomerismo , ChinaRESUMO
Garciyunnanones A-R (1-18), eighteen undescribed caged polycyclic polyprenylated acylphloroglucinols, two undescribed biogenetic congeners (19-20), and nineteen known analogues (21-39), were isolated from the stem barks of Garcinia yunnanensis Hu. All of these isolates are decorated with a C-5 lavandulyl substituent. Their structures and absolute configurations were confirmed by HRESIMS, 1D & 2D NMR spectroscopic analysis, quantum chemical calculations of electronic circular dichroism data, and single-crystal X-ray diffraction analysis. The X-ray crystallographic data of ten isolated caged compounds ascertained the absolute configuration of C-23 in the lavandulyl as S. The cytotoxicity on three cancer cell lines and the anti-nonalcoholic steatohepatitis activity of the isolates were tested. In a free fatty acid-induced L02 cell model, compounds 33 and 39 decreased intracellular lipid accumulation significantly.
Assuntos
Antineoplásicos Fitogênicos , Garcinia , Floroglucinol , Garcinia/química , Humanos , Floroglucinol/química , Floroglucinol/farmacologia , Floroglucinol/isolamento & purificação , Antineoplásicos Fitogênicos/farmacologia , Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/isolamento & purificação , Estrutura Molecular , Ensaios de Seleção de Medicamentos Antitumorais , Linhagem Celular Tumoral , Modelos Moleculares , Relação Estrutura-Atividade , Proliferação de Células/efeitos dos fármacos , Casca de Planta/químicaRESUMO
Medicinal compounds from plants include bicyclo[3.3.1]nonane derivatives, the majority of which are polycyclic polyprenylated acylphloroglucinols (PPAPs). Prototype molecules are hyperforin, the antidepressant constituent of St. John's wort, and garcinol, a potential anticancer compound. Their complex structures have inspired innovative chemical syntheses, however, their biosynthesis in plants is still enigmatic. PPAPs are divided into two subclasses, named type A and B. Here we identify both types in Hypericum sampsonii plants and isolate two enzymes that regiodivergently convert a common precursor to pivotal type A and B products. Molecular modelling and substrate docking studies reveal inverted substrate binding modes in the two active site cavities. We identify amino acids that stabilize these alternative binding scenarios and use reciprocal mutagenesis to interconvert the enzymatic activities. Our studies elucidate the unique biochemistry that yields type A and B bicyclo[3.3.1]nonane cores in plants, thereby providing key building blocks for biotechnological efforts to sustainably produce these complex compounds for preclinical development.
Assuntos
Hypericum , Hypericum/metabolismo , Hypericum/genética , Hypericum/química , Compostos Bicíclicos com Pontes/metabolismo , Compostos Bicíclicos com Pontes/química , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Simulação de Acoplamento Molecular , Floroglucinol/metabolismo , Floroglucinol/análogos & derivados , Floroglucinol/química , Alcanos/metabolismo , Alcanos/química , Domínio Catalítico , Terpenos/metabolismo , Terpenos/química , Modelos MolecularesRESUMO
Patumantanes A-D (1-4), four new seco-polycyclic polyprenylated acylphloroglucinols (PPAPs) were isolated from Hypericum patulum. Patumantane A (1) was an unprecedented 1,2-seco-homoadamantane-type PPAP bearing a new 3,7-dioxatetracyclo[7.7.0.01,6.111,15]heptadecane architecture based on a 6/7/5/6 ring system. Patumantane B (2) was a unique 1,9-seco-adamantane-type PPAP with a tricyclo[4.4.4.0.02,12]tridecane core formed by a 6/6/6 carbon skeleton, and the further breakage between C-5 and C-9 decorated patumantane C (3) with the 9-nor-adamantane skeleton. More importantly, compounds 2 and 3 exhibited moderate immunosuppressive activity on Con A-induced T-lymphocyte proliferation in vitro, with IC50 values of 5.6 ± 1.2 and 11.2 ± 1.2 µM, respectively.
Assuntos
Hypericum , Floroglucinol , Hypericum/química , Floroglucinol/química , Floroglucinol/farmacologia , Floroglucinol/análogos & derivados , Floroglucinol/isolamento & purificação , Humanos , Estrutura Molecular , Carbono/química , Proliferação de Células/efeitos dos fármacosRESUMO
St. John's wort (SJW) extract, a herbal medicine with antidepressant effects, is a potent inducer of intestinal and/or hepatic cytochrome P450 (CYP) enzymes and P-glycoprotein (P-gp), which can cause clinically relevant drug interactions. It is currently not known whether SJW can also induce P-gp activity at the human blood-brain barrier (BBB), which may potentially lead to decreased brain exposure and efficacy of certain central nervous system (CNS)-targeted P-gp substrate drugs. In this study, we used a combination of positron emission tomography (PET) imaging and cocktail phenotyping to gain a comprehensive picture on the effect of SJW on central and peripheral P-gp and CYP activities. Before and after treatment of healthy volunteers (n = 10) with SJW extract with a high hyperforin content (3-6%) for 12-19 days (1800 mg/day), the activity of P-gp at the BBB was assessed by means of PET imaging with the P-gp substrate [11C]metoclopramide and the activity of peripheral P-gp and CYPs was assessed by administering a low-dose phenotyping cocktail (caffeine, omeprazole, dextromethorphan, and midazolam or fexofenadine). SJW significantly increased peripheral P-gp, CYP3A, and CYP2C19 activity. Conversely, no significant changes in the peripheral metabolism, brain distribution, and P-gp-mediated efflux of [11C]metoclopramide across the BBB were observed following the treatment with SJW extract. Our data suggest that SJW does not lead to significant P-gp induction at the human BBB despite its ability to induce peripheral P-gp and CYPs. Simultaneous intake of SJW with CNS-targeted P-gp substrate drugs is not expected to lead to P-gp-mediated drug interactions at the BBB.
Assuntos
Barreira Hematoencefálica , Hypericum , Floroglucinol , Floroglucinol/análogos & derivados , Extratos Vegetais , Tomografia por Emissão de Pósitrons , Terfenadina/análogos & derivados , Terpenos , Humanos , Hypericum/química , Barreira Hematoencefálica/metabolismo , Barreira Hematoencefálica/efeitos dos fármacos , Floroglucinol/farmacocinética , Floroglucinol/farmacologia , Floroglucinol/administração & dosagem , Extratos Vegetais/farmacologia , Extratos Vegetais/administração & dosagem , Extratos Vegetais/farmacocinética , Masculino , Adulto , Tomografia por Emissão de Pósitrons/métodos , Terpenos/farmacologia , Terpenos/farmacocinética , Terpenos/metabolismo , Feminino , Adulto Jovem , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Compostos Bicíclicos com Pontes/farmacologia , Compostos Bicíclicos com Pontes/farmacocinética , Compostos Bicíclicos com Pontes/administração & dosagem , Terfenadina/farmacocinética , Terfenadina/administração & dosagem , Terfenadina/farmacologia , Sistema Enzimático do Citocromo P-450/metabolismo , Voluntários SaudáveisRESUMO
Hyperforatums A-D (1-4), four new polyprenylated acylphloroglucinols, together with 13 known compounds were isolated and identified from the aerial parts of Hypericum perforatum L. (St. John's wort). Their structures were confirmed with a comprehensive analysis comprising spectroscopic methods, including 1D and 2D NMR, HRESIMS, and electronic circular dichroism (ECD) calculations. Hyperforatum A featured an unusual chromene-1,4-dione bicyclic system, and hyperforatums B and C were two rare monocyclic PPAPs with five-membered furanone cores. Compound 1 exhibited a moderate inhibition effect on NO production in BV-2 microglial cells stimulated by LPS.
Assuntos
Hypericum , Floroglucinol , Hypericum/química , Floroglucinol/química , Floroglucinol/farmacologia , Floroglucinol/isolamento & purificação , Floroglucinol/análogos & derivados , Estrutura Molecular , Camundongos , Microglia/efeitos dos fármacos , Microglia/metabolismo , Animais , Óxido Nítrico/metabolismo , Óxido Nítrico/biossíntese , Linhagem Celular , Espectroscopia de Ressonância Magnética , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Lipopolissacarídeos/farmacologiaRESUMO
Pregnane X receptor (PXR) has been considered as a promising therapeutic target for cholestasis due to its crucial regulation in bile acid biosynthesis and metabolism. To search promising natural PXR agonists, the PXR agonistic activities of five traditional Chinese medicines (TCMs) with hepatoprotective efficacy were assayed, and Hypericum japonicum as the most active one was selected for subsequent phytochemical investigation, which led to the isolation of eight nonaromatic acylphloroglucinol-terpenoid adducts including seven new compounds (1 - 4, 5a, 5b and 6). Their structures including absolute configurations were determined by comprehensive spectroscopic, computational and X-ray diffraction analysis. Meanwhile, the PXR agonistic activities of aplenty compounds were evaluated via dual-luciferase reporter assay, RT-qPCR and immunofluorescence. Among them, compounds 1 - 4 showed more potent activity than the positive drug rifampicin. Furthermore, the molecular docking revealed that 1 - 4 were docked well on the PXR ligand binding domain and formed hydrogen bonds with amino acid residues Gln285, Ser247 and His409. This investigation revealed that H. japonicum may serve as a rich source of natural PXR agonists.
Assuntos
Hypericum , Simulação de Acoplamento Molecular , Floroglucinol , Receptor de Pregnano X , Hypericum/química , Receptor de Pregnano X/agonistas , Receptor de Pregnano X/metabolismo , Humanos , Floroglucinol/farmacologia , Floroglucinol/química , Floroglucinol/análogos & derivados , Relação Estrutura-Atividade , Estrutura Molecular , Terpenos/farmacologia , Terpenos/química , Terpenos/isolamento & purificação , Relação Dose-Resposta a Droga , Descoberta de Drogas , Células Hep G2RESUMO
This work explores the use of 2-nitrophloroglucinol (2-NPG) as a matrix for quantitative analysis of the fungicide Pyrimethanil (PYM) in strawberries using matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) and imaging. 2-NPG was selected for PYM analysis for optimum sensitivity and precision compared to common matrices α-cyano-4-hydroxylcinnamic acid (CHCA) and 2,5-dihydroxybenzoic acid (DHB). PYM-sprayed strawberries were frozen 0, 1, 3, and 4 days after treatment and sectioned for MALDI imaging. The remaining part of each strawberry was processed using quick easy cheap effective rugged and safe (QuEChERS) extraction and analyzed by MALDI-MS and ultraperformance liquid chromatography multireaction-monitoring (UPLC-MRM). MALDI-MS showed comparable performance to UPLC-MRM in calibration, LOD/LOQ, matrix effect, and recovery, with the benefit of fast analysis. The MALDI imaging results demonstrated that PYM progressively penetrated the interior of the strawberry over time and the PYM concentration on tissue measured by MALDI imaging correlated linearly with MALDI-MS and UPLC-MRM measurements and accounts for 79% MALDI-MS and 85% UPLC-MRM values on average. Additionally, quartz crystal microbalance (QCM) was introduced as a new approach to determine strawberry tissue mass per area for MALDI imaging absolute quantitation with sensitive, direct, and localized measurements. This work demonstrates the first example of absolute quantitative MALDI imaging of pesticides in a heterogeneous plant tissue. The novel use of the 2-NPG matrix in quantitative MALDI-MS and imaging could be applied to other analytes, and the new QCM tissue mass per area method is potentially useful for quantitative MALDI imaging of heterogeneous tissues in general.
Assuntos
Fragaria , Fungicidas Industriais , Pirimidinas , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Fragaria/química , Fungicidas Industriais/análise , Pirimidinas/análise , Pirimidinas/química , Floroglucinol/análise , Floroglucinol/química , Floroglucinol/análogos & derivados , Limite de Detecção , Cromatografia Líquida de Alta Pressão/métodosRESUMO
AIM: To explore the neuroprotective potential of hyperforin and elucidate its underlying molecular mechanisms involved in its therapeutic effects against vascular cognitive impairment (VCI). METHODS: The active compounds and possible targets of Hypericum perforatum L. that may be effective against VCI were found by network pharmacology in this research. We utilized bilateral common carotid artery occlusion (BCCAO) surgery to induce a VCI mouse model. Morris water maze (MWM) and Y-maze tests were used to assess VCI mice's cognitive abilities following treatment with hyperforin. To evaluate white matter lesions (WMLs), we utilized Luxol fast blue (LFB) stain and immunofluorescence (IF). Neuroinflammation was assessed using IF, western blot (WB), and enzyme-linked immunosorbent assay (ELISA). The effects of hyperforin on microglia were investigated by subjecting the BV2 microglial cell line to oxygen-glucose deprivation/reperfusion (OGD/R) stimulation. The expressions of VEGFR2 , p-SRC, SRC, VEGFA, and inflammatory markers including IL-10, IL-1ß, TNF-α, and IL-6 were subsequently assessed. RESULTS: The VEGFR2 /SRC signaling pathway is essential for mediating the protective properties of hyperforin against VCI according to network pharmacology analysis. In vivo findings demonstrated that hyperforin effectively improved BCCAO-induced cognitive impairment. Furthermore, staining results showed that hyperforin attenuated WMLs and reduced microglial activation in VCI mice. The hyperforin treatment group's ELISA results revealed a substantial decrease in IL-1ß, IL-6, and TNF-α levels. According to the results of in vitro experiments, hyperforin decreased the release of pro-inflammatory mediators (TNF-α, IL-6, and IL-1ß) and blocked microglial M1-polarization by modulating the VEGFR2 /SRC signaling pathway. CONCLUSION: Hyperforin effectively modulated microglial M1 polarization and neuroinflammation by inhibiting the VEGFR2 /SRC signaling pathways, thereby ameliorating WMLs and cognitive impairment in VCI mice.