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1.
Curr Microbiol ; 81(6): 166, 2024 May 09.
Article in English | MEDLINE | ID: mdl-38724665

ABSTRACT

Many regulatory genes that affect cellular development in Streptomyces, such as the canonical bld genes, have already been identified. However, in this study, we identified sven_5003 in Streptomyces venezuelae as a major new developmental regulatory gene, the deletion of which leads to a bald phenotype, typical of bld mutants, under multiple growth conditions. Our data indicated that disruption of sven_5003 also has a differential impact on the production of the two antibiotics jadomycin and chloramphenicol. Enhanced production of jadomycin but reduced production of chloramphenicol were detected in our sven_5003 mutant strain (S. venezuelae D5003). RNA-Seq analysis indicated that SVEN_5003 impacts expression of hundreds of genes, including genes involved in development, primary and secondary metabolism, and genes of unknown function, a finding confirmed by real-time PCR analysis. Transcriptional analysis indicated that sven_5003 is an auto-regulatory gene, repressing its own expression. Despite the evidence indicating that SVEN_5003 is a regulatory factor, a putative DNA-binding domain was not predicted from its primary amino acid sequence, implying an unknown regulatory mechanism by SVEN_5003. Our findings revealed that SVEN_5003 is a pleiotropic regulator with a critical role in morphological development in S. venezuelae.


Subject(s)
Anti-Bacterial Agents , Bacterial Proteins , Gene Expression Regulation, Bacterial , Streptomyces , Streptomyces/genetics , Streptomyces/metabolism , Streptomyces/growth & development , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Anti-Bacterial Agents/pharmacology , Chloramphenicol/pharmacology , Isoquinolines/metabolism
2.
BMC Plant Biol ; 24(1): 276, 2024 Apr 11.
Article in English | MEDLINE | ID: mdl-38605285

ABSTRACT

BACKGROUND: Stephania kwangsiensis Lo (Menispermaceae) is a well-known Chinese herbal medicine, and its bulbous stems are used medicinally. The storage stem of S. kwangsiensis originated from the hypocotyls. To date, there are no reports on the growth and development of S. kwangsiensis storage stems. RESULTS: The bulbous stem of S. kwangsiensis, the starch diameter was larger at the stable expanding stage (S3T) than at the unexpanded stage (S1T) or the rapidly expanding stage (S2T) at the three different time points. We used ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) and Illumina sequencing to identify key genes involved in bulbous stem development. A large number of differentially accumulated metabolites (DAMs) and differentially expressed genes (DEGs) were identified. Based on the differential expression profiles of the metabolites, alkaloids, lipids, and phenolic acids were the top three differentially expressed classes. Compared with S2T, significant changes in plant signal transduction and isoquinoline alkaloid biosynthesis pathways occurred at both the transcriptional and metabolic levels in S1T. In S2T compared with S3T, several metabolites involved in tyrosine metabolism were decreased. Temporal analysis of S1T to S3T indicated the downregulation of phenylpropanoid biosynthesis, including lignin biosynthesis. The annotation of key pathways showed an up-down trend for genes and metabolites involved in isoquinoline alkaloid biosynthesis, whereas phenylpropanoid biosynthesis was not completely consistent. CONCLUSIONS: Downregulation of the phenylpropanoid biosynthesis pathway may be the result of carbon flow into alkaloid synthesis and storage of lipids and starch during the development of S. kwangsiensis bulbous stems. A decrease in the number of metabolites involved in tyrosine metabolism may also lead to a decrease in the upstream substrates of phenylpropane biosynthesis. Downregulation of lignin synthesis during phenylpropanoid biosynthesis may loosen restrictions on bulbous stem expansion. This study provides the first comprehensive analysis of the metabolome and transcriptome profiles of S. kwangsiensis bulbous stems. These data provide guidance for the cultivation, breeding, and harvesting of S. kwangsiensis.


Subject(s)
Alkaloids , Plants, Medicinal , Stephania , Stephania/chemistry , Stephania/metabolism , Plants, Medicinal/metabolism , Chromatography, Liquid/methods , Lignin/metabolism , Tandem Mass Spectrometry , Plant Breeding , Gene Expression Profiling , Transcriptome , Alkaloids/metabolism , Starch/metabolism , Isoquinolines/metabolism , Tyrosine/metabolism , Lipids , Gene Expression Regulation, Plant
3.
Chem Biodivers ; 21(4): e202301865, 2024 Apr.
Article in English | MEDLINE | ID: mdl-38415909

ABSTRACT

In this study, phytochemical and biological activity studies supported by docking were carried out on a species of the genus Glaucium, a repository of isoquinoline alkaloids. The GC-MS (Gas Chromatography-Mass Spectrometry) method is used to characterize the isoquinoline alkaloids of Glaucium flavum Crantz. (Papaveraceae). G. flavum was collected from seven different regions of Türkiye (Antalya, Urla-Izmir, Mordogan-Izmir, Mugla, Assos-Canakkale, Karabiga-Canakkale, Giresun) and totally 17 compounds were detected by GC-MS. Glaucine was found to be the major constituent in the sample collected from Mugla, whereas isocorydine was recorded to be the principal alkaloid in other samples. Further fractionation studies on G. flavum collected from Antalya province in Southwestern Türkiye, yielded five major alkaloids (isocorydine 1, dihydrosanguinarine 2, glaucine 3, dehydroglaucine 4, protopine 5) which were characterized by spectroscopic methods. Anticholinesterase activities of the extracts and isolated alkaloids were also tested by in vitro Ellman method. The isolated compounds were also analyzed by a molecular docking technique to determine the binding orientations in the gorge of the active site of acetylcholinesterase (AChE) and a homology model of butyrylcholinesterase (BuChE). This is the first comparative investigation of the phytochemical composition and biodiversity of Glaucium flavum species growing in Türkiye.


Subject(s)
Alkaloids , Antineoplastic Agents , Papaveraceae , Cholinesterase Inhibitors/pharmacology , Cholinesterase Inhibitors/metabolism , Butyrylcholinesterase/metabolism , Molecular Docking Simulation , Acetylcholinesterase/metabolism , Alkaloids/chemistry , Isoquinolines/pharmacology , Isoquinolines/metabolism , Antineoplastic Agents/metabolism , Papaveraceae/chemistry , Papaveraceae/metabolism , Phytochemicals/metabolism , Plant Extracts/chemistry
4.
Genes (Basel) ; 14(12)2023 Dec 18.
Article in English | MEDLINE | ID: mdl-38137054

ABSTRACT

Coptis chinensis is a perennial herb of the Ranunculaceae family. The isoquinoline alkaloid is the main active component of C. chinensis, mainly exists in its rhizomes and has high clinical application potential. The in vitro synthesis of isoquinoline alkaloids is difficult because their structures are complex; hence, plants are still the main source of them. In this study, two-year and four-year rhizomes of C. chinensis were selected to investigate the effect of growth years on the accumulation of isoquinoline alkaloids. Two-year and four-year C. chinensis were selected for metabolomics detection and transcriptomic analysis. A total of 413 alkaloids were detected by metabolomics analysis, of which 92 were isoquinoline alkaloids. (S)-reticuline was a significantly different accumulated metabolite of the isoquinoline alkaloids biosynthetic pathway in C. chinensis between the two groups. The results of transcriptome analysis showed that a total of 464 differential genes were identified, 36 of which were associated with the isoquinoline alkaloid biosynthesis pathway of C. chinensis. Among them, 18 genes were correlated with the content of important isoquinoline alkaloids. Overall, this study provided a comprehensive metabolomic and transcriptomic analysis of the rapid growth stage of C. chinensis rhizome from the perspective of growth years. It brought new insights into the biosynthetic pathway of isoquinoline alkaloids and provided information for utilizing biotechnology to improve their contents in C. chinensis.


Subject(s)
Alkaloids , Coptis , Coptis chinensis , Transcriptome , Coptis/genetics , Coptis/chemistry , Coptis/metabolism , Alkaloids/genetics , Alkaloids/metabolism , Metabolome , Isoquinolines/metabolism
5.
Ann Nucl Med ; 37(2): 108-120, 2023 Feb.
Article in English | MEDLINE | ID: mdl-36411357

ABSTRACT

OBJECTIVE: A new tau PET tracer [18F]MK-6240 has been developed; however, its dosimetry and pharmacokinetics have been published only for a European population. This study investigated the safety, radiation dosimetry, pharmacokinetics and biodistribution of [18F]MK-6240 in Japanese elderly subjects. Also, the pattern and extent of brain retention of [18F]MK-6240 in Japanese healthy elderly subjects and patients with Alzheimer's disease (AD) were investigated. These Japanese results were compared with previous reports on non-Japanese. METHODS: Three healthy elderly subjects and three AD patients were enrolled. Dynamic whole-body PET scans were acquired for up to 232 min after starting injection of [18F]MK-6240 (370.4 ± 27.0 MBq) for the former, while a dynamic brain scan was performed from 0 to 75 min post injection for the latter. For both groups, brain PET scans were conducted from 90 to 110 min post injection. Sequential venous blood sampling was performed to measure the radioactivity concentration in the whole blood and plasma as well as the percentages of parent [18F]MK-6240 and radioactive metabolites in plasma. Organ doses and effective doses were estimated using the OLINDA Ver.2 software. Standardized uptake value ratios (SUVRs) and distribution volume ratios (DVRs) by Logan reference tissue model (LRTM) were measured in eight brain regions using the cerebellar cortex as the reference. Blood tests, urine analysis, vital signs and electrocardiography were performed for safety assessments. RESULTS: No adverse events were observed. The highest radiation doses were received by the gallbladder (257.7 ± 74.9 µGy/MBq) and the urinary bladder (127.3 ± 11.7 µGy/MBq). The effective dose was 26.8 ± 1.4 µSv/MBq. The parent form ([18F]MK-6240) was metabolized quickly and was less than 15% by 35 min post injection. While no obvious accumulation was found in the brain of healthy subjects, focal accumulation of [18F]MK-6240 was observed in the cerebral cortex of AD patients. Regional SUVRs of the focal lesions in AD patients increased gradually over time, and the difference of SUVRs between healthy subjects and AD patients became large and stable at 90 min after injection. High correlations of SUVR and DVR were observed (p < 0.01). CONCLUSION: The findings supported safety and efficacy of [18F]MK-6240 as a tau PET tracer for Japanese populations. Even though the number of subjects was limited, the radiation dosimetry profiles, pharmacokinetics, and biodistribution of [18F]MK-6240 were consistent with those for non-Japanese populations. TRIAL REGISTRATION: Japan Pharmaceutical Information Center ID, JapicCTI-194972.


Subject(s)
Alzheimer Disease , Humans , Aged , Alzheimer Disease/diagnostic imaging , Alzheimer Disease/metabolism , Tissue Distribution , Radiometry , Isoquinolines/metabolism , Positron-Emission Tomography/methods
6.
Biomaterials ; 290: 121819, 2022 11.
Article in English | MEDLINE | ID: mdl-36209579

ABSTRACT

The faithful engineering of complex human tissues such as the bone/soft tissue/mineralized tissue interface in periodontal tissues requires innovative molecular cues in conjunction with tailored scaffolds. To address the loss of periodontal bone and connective tissues following periodontal disease, we have generated a polydopamine and collagen coated electrospun PLGA-PCL (PP) scaffold enriched with the small molecule mediator PFI-2 (PP-PFI-pDA-COL-PFI). In vitro 3D studies using PDL progenitors revealed that the PP-PFI-pDA-COL-PFI scaffold substantially enhanced Alizarin Red staining, increased Ca/P ratios 4-fold, and stimulated cell proliferation more than 12-fold compared to PP-controls, suggestive of its potential for mineralized tissue engineering. When applied in our experimental periodontitis model, the PP-PFI-pDA-COL-PFI scaffold resulted in a substantial 34% reduction in alveolar bone defect height, a 25% root-length gain in periodontal attachment, and the formation of highly ordered regenerated acellular cementum twice as thick as in controls. Explaining the mechanism of PFI-2 mineralized tissue regeneration in periodontal tissues, PFI-2 inhibited SETD7-mediated ß-Catenin protein methylation and increased ß-Catenin nuclear localization. Together, dual-level PFI-2 incorporation into a degradable, dopamine/collagen coated PLGA/PCL scaffold backbone resulted in the regeneration of the tripartite periodontal complex with unprecedented fidelity, including periodontal attachment and new formation of mineralized tissues in inflamed periodontal environments.


Subject(s)
Periodontal Ligament , Tissue Scaffolds , Humans , Isoquinolines/metabolism , Collagen/metabolism , Polylactic Acid-Polyglycolic Acid Copolymer , Histone-Lysine N-Methyltransferase/metabolism
7.
Int J Mol Sci ; 23(18)2022 Sep 17.
Article in English | MEDLINE | ID: mdl-36142812

ABSTRACT

Pinellia ternata (Thunb.) Druce is a traditional medicinal plant containing a variety of alkaloids, which are important active ingredients. Brassinolide (BR) is a plant hormone that regulates plant response to environmental stress and promotes the accumulation of secondary metabolites in plants. However, the regulatory mechanism of BR-induced alkaloid accumulation in P. ternata is not clear. In this study, we investigated the effects of BR and BR biosynthesis inhibitor (propiconazole, Pcz) treatments on alkaloid biosynthesis in the bulbil of P. ternata. The results showed that total alkaloid content and bulbil yield was enhanced by 90.87% and 29.67% under BR treatment, respectively, compared to the control. We identified 818 (476 up-regulated and 342 down-regulated) and 697 (389 up-regulated and 308 down-regulated) DEGs in the BR-treated and Pcz-treated groups, respectively. Through this annotated data and the Kyoto encyclopedia of genes and genomes (KEGG), the expression patterns of unigenes involved in the ephedrine alkaloid, tropane, piperidine, pyridine alkaloid, indole alkaloid, and isoquinoline alkaloid biosynthesis were observed under BR and Pcz treatments. We identified 11, 8, 2, and 13 unigenes in the ephedrine alkaloid, tropane, piperidine, and pyridine alkaloid, indole alkaloid, and isoquinoline alkaloid biosynthesis, respectively. The expression levels of these unigenes were increased by BR treatment and were decreased by Pcz treatment, compared to the control. The results provided molecular insight into the study of the molecular mechanism of BR-promoted alkaloid biosynthesis.


Subject(s)
Alkaloids , Pinellia , Alkaloids/metabolism , Brassinosteroids , Ephedrine , Gene Expression Profiling , Isoquinolines/metabolism , Pinellia/genetics , Piperidines/metabolism , Plant Growth Regulators/metabolism , Plant Growth Regulators/pharmacology , Pyridines/metabolism , Steroids, Heterocyclic , Transcriptome , Tropanes
8.
J Integr Neurosci ; 21(2): 71, 2022 Mar 23.
Article in English | MEDLINE | ID: mdl-35364659

ABSTRACT

As a catechol isoquinoline, salsolinol (Sal) is widely distributed in mammalian brains, and is increased in the cerebrospinal fluid (CSF) and urine of Parkinsonian patients. Sal can be metabolized to N-methyl-salsolinol (NM-Sal), an MPP+-like neurotoxin, and impairs the function of dopaminergic neurons, causing the clinical symptoms of Parkinson's disease (PD). Sal synthase, which catalyzes the production of Sal from dopamine and acetaldehyde, may be the important enzyme in the metabolism of catechol isoquinolines (CTIQs). Previously, our work demonstrated the existence of Sal synthase in rat brain and identified its amino acid sequence. However, the biological function of Sal synthase has not been thoroughly explored, especially its role in dopaminergic neuronal degeneration. In this study, we tried to clarify the catalytic role of Sal synthase in the formation of CTIQs which are endogenous neurotoxins in the mammalian brain. Furthermore, the cytotoxicity of Sal synthase was also observed in dopaminergic PC12 cells. The results demonstrated that Sal synthase overexpression can increase the level of Sal and NM-Sal, and ultimately cause mitochondria damage and apoptosis.


Subject(s)
Dopamine , Tetrahydroisoquinolines , Animals , Dopamine/chemistry , Dopamine/metabolism , Humans , Isoquinolines/chemistry , Isoquinolines/metabolism , Mammals/metabolism , PC12 Cells , Rats
9.
ACS Chem Biol ; 17(3): 598-608, 2022 03 18.
Article in English | MEDLINE | ID: mdl-35253423

ABSTRACT

Isoquinoline alkaloids are a large class of natural products with a broad range of biological activities, including antimicrobial, antitumor, antileukemic and anti-inflammatory properties. Although mostly found in plants, isoquinolines can also be found in the extracts of bacterial and fungal cultures. Regardless of the origin, most of the reported biosynthetic routes for isoquinolines use tyrosine as a main biosynthetic precursor. Here, we report the identification of a new biosynthetic pathway for production of isoquinolinequinone alkaloid mansouramycin D in Streptomyces albus Del14. Using feeding, mass spectrometry, and nuclear magnetic resonance spectroscopy, we demonstrate that tryptophan serves instead of tyrosine as a main mansouramycin biosynthetic precursor. The biosynthetic genes were identified in the chromosome of the strain by using gene inactivation and heterologous expression. Insights into the biosynthesis of mansouramycins are also presented.


Subject(s)
Alkaloids , Isoquinolines , Alkaloids/chemistry , Biosynthetic Pathways/genetics , Isoquinolines/metabolism , Tyrosine/metabolism
10.
Biochem Pharmacol ; 197: 114939, 2022 03.
Article in English | MEDLINE | ID: mdl-35114188

ABSTRACT

Kidney injury often causes anemia due to a lack of production of the erythroid growth factor erythropoietin (EPO) in the kidneys. Roxadustat is one of the first oral medicines inducing EPO production in patients with renal anemia by activating hypoxia-inducible factors (HIFs), which are activators of EPO gene expression. In this study, to develop prodrugs of roxadustat with improved permeability through cell membrane, we investigated the effects of 8 types of esterification on the pharmacokinetics and bioactivity of roxadustat using Hep3B hepatoma cells that HIF-dependently produce EPO. Mass spectrometry of cells incubated with the esterified roxadustat derivatives revealed that the designed compounds were deesterified after being taken up by cells and showed low cytotoxicity compared to the original compound. Esterification prolonged the effective duration of roxadustat with respect to EPO gene induction and HIF activation in cells transiently exposed to the compounds. In the kidneys and livers of mice, both of which are unique sites of EPO production, a majority of the methyl-esterified roxadustat was deesterified within 6 h after drug administration. The deesterified roxadustat derivative was continuously detectable in plasma and urine for at least 48 h after administration, while the administered compound became undetectable 24 h after administration. Additionally, we confirmed that methyl-esterified roxadustat activated erythropoiesis in mice by inducing Epo mRNA expression exclusively in renal interstitial cells, which have intrinsic EPO-producing potential. These data suggest that esterification could lead to the development of roxadustat prodrugs with improvements in cell membrane permeability, effective duration and cytotoxicity.


Subject(s)
Basic Helix-Loop-Helix Transcription Factors/metabolism , Cell Survival/drug effects , Glycine/analogs & derivatives , Hypoxia-Inducible Factor-Proline Dioxygenases/metabolism , Intracellular Membranes/metabolism , Isoquinolines/metabolism , Isoquinolines/pharmacology , Animals , Basic Helix-Loop-Helix Transcription Factors/agonists , Cell Survival/physiology , Dose-Response Relationship, Drug , Esterification/drug effects , Esterification/physiology , Glycine/metabolism , Glycine/pharmacology , Humans , Intracellular Membranes/drug effects , Male , Mice , Mice, Inbred C57BL , Mice, Inbred ICR , Time Factors , Treatment Outcome , Tumor Cells, Cultured
11.
Microb Cell Fact ; 21(1): 9, 2022 Jan 10.
Article in English | MEDLINE | ID: mdl-35012550

ABSTRACT

The biosynthetic pathways for the fungal polyketides bikaverin and bostrycoidin, from Fusarium verticillioides and Fusarium solani respectively, were reconstructed and heterologously expressed in S. cerevisiae alongside seven different phosphopantetheinyl transferases (PPTases) from a variety of origins spanning bacterial, yeast and fungal origins. In order to gauge the efficiency of the interaction between the ACP-domains of the polyketide synthases (PKS) and PPTases, each were co-expressed individually and the resulting production of target polyketides were determined after 48 h of growth. In co-expression with both biosynthetic pathways, the PPTase from Fusarium verticillioides (FvPPT1) proved most efficient at producing both bikaverin and bostrycoidin, at 1.4 mg/L and 5.9 mg/L respectively. Furthermore, the remaining PPTases showed the ability to interact with both PKS's, except for a single PKS-PPTase combination. The results indicate that it is possible to boost the production of a target polyketide, simply by utilizing a more optimal PPTase partner, instead of the commonly used PPTases; NpgA, Gsp and Sfp, from Aspergillus nidulans, Brevibacillus brevis and Bacillus subtilis respectively.


Subject(s)
Bacterial Proteins/metabolism , Fusarium/enzymology , Polyketide Synthases/metabolism , Transferases (Other Substituted Phosphate Groups)/metabolism , Xanthones/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Biosynthetic Pathways , Cloning, Molecular , Fusarium/genetics , Isoquinolines/metabolism , Models, Molecular , Polyketide Synthases/chemistry , Polyketide Synthases/genetics , Protein Domains , Protein Structure, Tertiary , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Transferases (Other Substituted Phosphate Groups)/chemistry , Transferases (Other Substituted Phosphate Groups)/genetics
12.
Alzheimers Dement ; 18(2): 272-282, 2022 02.
Article in English | MEDLINE | ID: mdl-34057284

ABSTRACT

INTRODUCTION: Positron emission tomography (PET) imaging for neurofibrillary tau allows investigation of the in vivo spatiotemporal progression of Alzheimer's disease (AD) pathology. We evaluated the suitability of 18 F-MK-6240 in a clinical sample and determined the relationships among 18 F-MK-6240 binding, age, cognition, and cerebrospinal fluid (CSF)-based AD biomarkers. METHODS: Participants (n = 101, 72 ± 9 years, 52% women) underwent amyloid PET, tau PET, structural T1-weighted magnetic resonance imaging, and neuropsychological evaluation. Twenty-one participants had lumbar puncture for CSF measurement of amyloid beta (Aß)42 , tau, and phosphorylated tau (p-tau). RESULTS: 18 F-MK-6240 recapitulated Braak staging and correlated with CSF tau and p-tau, normalized to Aß42 . 18 F-MK-6240 negatively correlated with age across Braak regions in amyloid-positive participants, consistent with greater tau pathology in earlier onset AD. Domain-specific, regional patterns of 18 F-MK-6240 binding were associated with reduced memory, executive, and language performance, but only in amyloid-positive participants. DISCUSSION: 18 F-MK-6240 can approximate Braak staging across the AD continuum and provide region-dependent insights into biomarker-based AD models.


Subject(s)
Alzheimer Disease , Cognitive Dysfunction , Aged , Aged, 80 and over , Alzheimer Disease/pathology , Amyloid beta-Peptides/metabolism , Biomarkers , Cognitive Dysfunction/diagnostic imaging , Cognitive Dysfunction/metabolism , Female , Humans , Isoquinolines/metabolism , Male , Middle Aged , Positron-Emission Tomography/methods , tau Proteins/cerebrospinal fluid
13.
ChemMedChem ; 17(3): e202100658, 2022 02 04.
Article in English | MEDLINE | ID: mdl-34797951

ABSTRACT

In our constant search for new successors of agomelatine, we report herein a new series of compounds resulting from bioisosteric modulation of the naphthalene ring. The isoquinoline and tetrahydroisoquinoline derivatives were synthesized and pharmacologically evaluated. This isosteric replacement of the naphthalene group of agomelatine has led to potent agonist and partial agonist compounds with nanomolar melatonergic binding affinities. Overall, the presence of a nitrogen atom was accompanied with a decrease in the binding affinity toward both MT1 and MT2 and the loss of 5HT2C response, especially for tetrahydroisoquinoline in comparison with the parent compound. Interestingly, due to the presence of this nitrogen atom, a notable improvement in the pharmacokinetic properties was observed for all compounds.


Subject(s)
Isoquinolines/pharmacology , Receptors, Melatonin/agonists , Animals , Cells, Cultured , Cricetulus , Dose-Response Relationship, Drug , Humans , Isoquinolines/chemistry , Isoquinolines/metabolism , Ligands , Molecular Docking Simulation , Molecular Structure , Structure-Activity Relationship
14.
Fitoterapia ; 156: 105086, 2022 Jan.
Article in English | MEDLINE | ID: mdl-34798164

ABSTRACT

Two new isoquinoline alkaloids, cryptowrayines A (1) and B (2), along with one known pavine alkaloid (-)-12-hydroxyeschscholtzidine (3), were isolated from the twigs of Cryptocarya wrayi. The structures of new compounds were elucidated by extensive spectroscopic data analysis and electronic circular dichroism (ECD) calculations. Both compounds 1 and 2 exhibited moderate quinone reductase inducing activity in Hepa 1c1c7 cells.


Subject(s)
Alkaloids/isolation & purification , Cryptocarya/chemistry , Isoquinolines/isolation & purification , Alkaloids/chemistry , Alkaloids/metabolism , Glucosidases/antagonists & inhibitors , Glycoside Hydrolase Inhibitors/chemistry , Glycoside Hydrolase Inhibitors/isolation & purification , Glycoside Hydrolase Inhibitors/metabolism , Inhibitory Concentration 50 , Isoquinolines/chemistry , Isoquinolines/metabolism , Magnetic Resonance Spectroscopy , Molecular Structure , NAD(P)H Dehydrogenase (Quinone)/analysis , Optical Rotation
15.
Nat Commun ; 12(1): 7085, 2021 12 06.
Article in English | MEDLINE | ID: mdl-34873166

ABSTRACT

Antibiotic resistance is becoming one of the major crises, among which hydrolysis reaction is widely employed by bacteria to destroy the reactive pharmacophore. Correspondingly, antibiotic producer has canonically co-evolved this approach with the biosynthetic capability for self-resistance. Here we discover a self-defense strategy featuring with reductive inactivation of hemiaminal pharmacophore by short-chain dehydrogenases/reductases (SDRs) NapW and homW, which are integrated with the naphthyridinomycin biosynthetic pathway. We determine the crystal structure of NapW·NADPH complex and propose a catalytic mechanism by molecular dynamics simulation analysis. Additionally, a similar detoxification strategy is identified in the biosynthesis of saframycin A, another member of tetrahydroisoquinoline (THIQ) antibiotics. Remarkably, similar SDRs are widely spread in bacteria and able to inactive other THIQ members including the clinical anticancer drug, ET-743. These findings not only fill in the missing intracellular events of temporal-spatial shielding mode for cryptic self-resistance during THIQs biosynthesis, but also exhibit a sophisticated damage-control in secondary metabolism and general immunity toward this family of antibiotics.


Subject(s)
Bacteria/metabolism , Bacterial Proteins/metabolism , Biosynthetic Pathways , Molecular Dynamics Simulation , Tetrahydroisoquinolines/metabolism , Anti-Bacterial Agents/biosynthesis , Anti-Bacterial Agents/chemistry , Bacteria/genetics , Bacterial Proteins/genetics , Biocatalysis , Chromatography, High Pressure Liquid , Drug Resistance, Microbial/genetics , Humans , Isoquinolines/chemistry , Isoquinolines/metabolism , Mass Spectrometry/methods , Molecular Structure , NADP/chemistry , NADP/metabolism , Naphthyridines/chemistry , Naphthyridines/metabolism , Oxidation-Reduction , Oxidoreductases/genetics , Oxidoreductases/metabolism , Tetrahydroisoquinolines/chemistry
16.
Proc Natl Acad Sci U S A ; 118(51)2021 12 21.
Article in English | MEDLINE | ID: mdl-34903659

ABSTRACT

Benzylisoquinoline alkaloids (BIAs) are a diverse class of medicinal plant natural products. Nearly 500 dimeric bisbenzylisoquinoline alkaloids (bisBIAs), produced by the coupling of two BIA monomers, have been characterized and display a range of pharmacological properties, including anti-inflammatory, antitumor, and antiarrhythmic activities. In recent years, microbial platforms have been engineered to produce several classes of BIAs, which are rare or difficult to obtain from natural plant hosts, including protoberberines, morphinans, and phthalideisoquinolines. However, the heterologous biosyntheses of bisBIAs have thus far been largely unexplored. Here, we describe the engineering of yeast strains that produce the Type I bisBIAs guattegaumerine and berbamunine de novo. Through strain engineering, protein engineering, and optimization of growth conditions, a 10,000-fold improvement in the production of guattegaumerine, the major bisBIA pathway product, was observed. By replacing the cytochrome P450 used in the final coupling reaction with a chimeric variant, the product profile was inverted to instead produce solely berbamunine. Our highest titer engineered yeast strains produced 108 and 25 mg/L of guattegaumerine and berbamunine, respectively. Finally, the inclusion of two additional putative BIA biosynthesis enzymes, SiCNMT2 and NnOMT5, into our bisBIA biosynthetic strains enabled the production of two derivatives of bisBIA pathway intermediates de novo: magnocurarine and armepavine. The de novo heterologous biosyntheses of bisBIAs presented here provide the foundation for the production of additional medicinal bisBIAs in yeast.


Subject(s)
Benzylisoquinolines/metabolism , Isoquinolines/metabolism , Saccharomyces cerevisiae/metabolism , Alkaloids/biosynthesis , Biosynthetic Pathways , Cytochrome P-450 Enzyme System/genetics , Cytochrome P-450 Enzyme System/metabolism , Fermentation , Metabolic Engineering , Plant Proteins/genetics , Plant Proteins/metabolism , Protein Engineering , Racemases and Epimerases/genetics , Racemases and Epimerases/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Saccharomyces cerevisiae/genetics , Tetrahydroisoquinolines/chemistry , Tetrahydroisoquinolines/metabolism
17.
Pharm Res ; 38(10): 1663-1675, 2021 Oct.
Article in English | MEDLINE | ID: mdl-34647232

ABSTRACT

PURPOSE: Organic Anion Transporting Polypeptide 1B1 (OATP1B1) mediates hepatic influx and clearance of many drugs, including statins. The SLCO1B1 gene is highly polymorphic and its function-impairing variants can predispose patients to adverse effects. The effects of rare genetic variants of SLCO1B1 are mainly unexplored. We examined the impact of eight naturally occurring rare variants and the well-known SLCO1B1 c.521C > T (V174A) variant on in vitro transport activity, cellular localization and abundance. METHODS: Transport of rosuvastatin and 2,7-dichlorofluorescein (DCF) in OATP1B1 expressing HEK293 cells was measured to assess changes in activity of the variants. Immunofluorescence and confocal microscopy determined the cellular localization of OATP1B1 and LC-MS/MS based quantitative targeted absolute proteomics analysis quantified the amount of OATP1B1 in crude membrane fractions. RESULTS: All studied variants, with the exception of P336R, reduced protein abundance to varying degree. V174A reduced protein abundance the most, over 90% compared to wild type. Transport function was lost in G76E, V174A, L193R and R580Q variants. R181C decreased activity significantly, while T345M and L543W retained most of wild type OATP1B1 activity. P336R showed increased activity and H575L decreased the transport of DCF significantly, but not of rosuvastatin. Decreased activity was interrelated with lower absolute protein abundance in the studied variants. CONCLUSIONS: Transmembrane helices 2, 4 and 11 appear to be crucial for proper membrane localization and function of OATP1B1. Four of the studied variants were identified as loss-of-function variants and as such could make the individual harboring these variants susceptible to altered pharmacokinetics and adverse effects of substrate drugs.


Subject(s)
Hydroxymethylglutaryl-CoA Reductase Inhibitors/metabolism , Isoquinolines/metabolism , Liver-Specific Organic Anion Transporter 1/metabolism , Nucleotides/metabolism , Rosuvastatin Calcium/metabolism , Biological Transport , Drug Interactions , Gene Expression , HEK293 Cells , Humans , Liver , Liver-Specific Organic Anion Transporter 1/genetics , Mutation , Polymorphism, Genetic , Tandem Mass Spectrometry
18.
Sci Rep ; 11(1): 21191, 2021 10 27.
Article in English | MEDLINE | ID: mdl-34707100

ABSTRACT

The American cockroach, Periplaneta americana (L.), is able to highly survive in various complicated environments around the globe, and often considered as a pest. In contrast, billions of P. americana have been massively reared in China and extensively used as a medicinal insect, due to its function for preventing and treating ulceration and heart failure. Considering the possibility that microbiota-derived metabolites could be an effective source to identify promising candidate drugs, we attempted to establish a rapid method for simultaneous determination of gut microbiota metabolites from medicinal insects. In this study, network pharmacology approach and ultra-performance liquid chromatography (UPLC) technique were employed to reveal the potential pharmacological activity and dynamics variation of nitrogen-containing metabolites (NCMs) originated from the gut microbiota of breeding P. americana at different growth stages. A metabolites-targets-diseases network showed that NCMs are likely to treat diseases such as ulceration and cancer. The analysis of NCMs' content with the growth pattern of P. americana indicated that the content of NCMs declined with P. americana aging. Both principal component analysis and orthogonal partial least squares discriminant analysis suggested that 8-hydroxy-2-quinolinecarboxylic acid and 8-hydroxy-3,4-dihydro-2(1H)-quinolinone are the potential differential metabolic markers for discriminating between nymphs and adults of P. americana. Moreover, the developed UPLC method showed an excellent linearity (R2 > 0.999), repeatability (RSD < 2.6%), intra- and inter-day precisions (RSD < 2.2%), and recovery (95.5%-99.0%). Collectively, the study provides a valuable strategy for analyzing gut microbiota metabolites from insects and demonstrates the prospects for discovering novel drug candidates from the feces of P. americana.


Subject(s)
Biological Products/metabolism , Cockroaches/microbiology , Gastrointestinal Microbiome , Isoquinolines/metabolism , Animals , Biological Products/analysis , Carboxylic Acids/analysis , Carboxylic Acids/metabolism , Cockroaches/growth & development , Feces/chemistry , Feces/microbiology , Isoquinolines/analysis , Nitrogen/metabolism , Nymph/growth & development , Nymph/microbiology
19.
Toxins (Basel) ; 13(6)2021 05 25.
Article in English | MEDLINE | ID: mdl-34070644

ABSTRACT

Natural products display a large structural variation and different uses within a broad spectrum of industries. In this study, we investigate the influence of carbohydrates and nitrogen sources on the production and selectivity of production of four different polyketides produced by Fusarium solani, fusarubin, javanicin, bostrycoidin and anhydrofusarubin. We introduce four different carbohydrates and two types of nitrogen sources. Hereafter, a full factorial design was applied using combinations of three levels of sucrose and three levels of the two types of nitrogen. Each combination displayed different selectivity and production yields for all the compounds of interest. Response surface design was utilized to investigate possible maximum yields for the surrounding combinations of media. It was also shown that the maximum yields were not always the ones illustrating high selectivity, which is an important factor for making purification steps easier. We visualized the production over time for one of the media types, illustrating high yields and selectivity.


Subject(s)
Fusarium/metabolism , Naphthoquinones/metabolism , Polyketides/metabolism , Culture Media , Isoquinolines/metabolism , Nitrogen/metabolism , Sucrose/metabolism
20.
Article in English | MEDLINE | ID: mdl-34051306

ABSTRACT

The biological cascade of second messenger-cyclic adenosine monophosphate (cAMP) -as a molecular mechanism implicated in memory and learning regulation has captured the attention of neuroscientists worldwide. cAMP triggers its foremost effector, protein kinase A (PKA), resulting in the activation of innumerable downstream targets. Roflumilast (ROF), a phosphodiesterase 4 inhibitor, has demonstrated a greater efficiency in enhancing cAMP signaling in various neurological disorders. This study was conducted to identify various downstream targets of PKA as mechanistic tools through which ROF could hinder the progressive cognitive impairment following central streptozotocin (STZ) administration in mice. Animals were injected with STZ (3 mg/kg/i.c.v) once. Five hours later, mice received ROF (0.4 mg/kg) with or without the PKA inhibitor, H89, for 21 days. ROF highly preserved the structure of hippocampal neurons. It improved the ability of mice to develop short-term memories and retrieve spatial memories in Y-maze and Morris water maze tests, respectively. ROF enhanced the gene expression of ABCB1 transporters and pregnane X receptors (PXR), and hampered Aß accumulation in hippocampus. Simultaneously, it interfered with the processes of tau phosphorylation and nitration. This effect was associated with an upsurge in hippocampal arginase activity as well as a decline in glycogen synthase kinase-3ß activity, nitric oxide synthase (NOS) activity, and inducible NOS expression. Contrariwise, ROF's beneficial effects were utterly abolished by co-administration of H89. In conclusion, boosting PKA, by ROF, modulated PXR/ABCB1 expression and arginase/NOS activities to restrict the main post-translational modifications of tau, Aß deposition and, accordingly, cognitive deterioration of sporadic Alzheimer's disease.


Subject(s)
Aminopyridines/pharmacology , Amyloid beta-Peptides/metabolism , Benzamides/pharmacology , Cognitive Dysfunction , Isoquinolines/metabolism , Nitric Oxide/metabolism , Protein Kinase Inhibitors/metabolism , Sulfonamides/metabolism , Tauopathies/metabolism , ATP Binding Cassette Transporter, Subfamily B, Member 1 , Alzheimer Disease/metabolism , Animals , Antibiotics, Antineoplastic/administration & dosage , Cognitive Dysfunction/metabolism , Cognitive Dysfunction/prevention & control , Cyclopropanes/pharmacology , Disease Models, Animal , Hippocampus/metabolism , Male , Mice , Phosphodiesterase 4 Inhibitors/pharmacology , Streptozocin/administration & dosage
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