Low Molecular Weight Natural Products Research Articles

Direct pathway cloning and expression of the radiosumin biosynthetic gene cluster.

Radiosumins are a structurally diverse family of low molecular weight natural products that are produced by cyanobacteria and exhibit potent serine protease inhibition. Members of this family are dipeptides characterized by the presence of two similar non-proteinogenic amino acids. Here we used a comparative bioinformatic analysis to identify radiosumin biosynthetic gene clusters from the genomes of 13 filamentous cyanobacteria. We used direct pathway cloning to capture and express the entire 16.8 kb radiosumin biosynthetic gene cluster from Dolichospermum planctonicum UHCC 0167 in Escherichia coli. Bioinformatic analysis demonstrates that radiosumins represent a new group of chorismate-derived non-aromatic secondary metabolites. High-resolution liquid chromatography-mass spectrometry, nuclear magnetic resonance spectroscopy and chemical degradation analysis revealed that cyanobacteria produce a cocktail of novel radiosumins. We report the chemical structure of radiosumin D, an N-methyl dipeptide, containing a special Aayp (2-amino-3-(4-amino-2-cyclohexen-1-ylidene) propionic acid) with R configuration that differs from radiosumin A-C, an N-Me derivative of Aayp (Amyp) and two acetyl groups. Radiosumin C inhibits all three human trypsin isoforms at micromolar concentrations with preference for trypsin-1 and -3 (IC50 values from 1.7 μM to >7.2 μM). These results provide a biosynthetic logic to explore the genetic and chemical diversity of the radiosumin family and suggest that these natural products may be a source of drug leads for selective human serine proteases inhibitors.

Matching chemical properties to molecular biological activities opens a new perspective on l-ergothioneine.

l-ergothioneine is a low-molecular weight natural product, the chemical structure of which comprises oxygen-, nitrogen- and sulfur-containing functional groups. This gives l-ergothioneine specific physicochemical properties and allows a better understanding of its chemical reactivity, which is primarily due to the 2-thio-imidazole group. Here, a review is provided of how different modes of chemical reactivity account for the reported molecular biological activities of l-ergothioneine. By matching the physicochemical properties to the biological properties of l-ergothioneine, a new perspective of the function and the mode of action of this enigmatic molecule emerges.

Discovery of varlaxins, new aeruginosin-type inhibitors of human trypsins.

Low-molecular weight natural products display vast structural diversity and have played a key role in the development of novel therapeutics. Here we report the discovery of novel members of the aeruginosin family of natural products, which we named varlaxins. The chemical structures of varlaxins 1046A and 1022A were determined using a combination of mass spectrometry, analysis of one- and two-dimensional NMR spectra, and HPLC analysis of Marfey's derivatives. These analyses revealed that varlaxins 1046A and 1022A are composed of the following moieties: 2-O-methylglyceric acid 3-O-sulfate, isoleucine, 2-carboxy-6-hydroxyoctahydroindole (Choi), and a terminal arginine derivative. Varlaxins 1046A and 1022A differ in the cyclization of this arginine moiety. Interestingly, an unusual α-D-glucopyranose moiety derivatized with two 4-hydroxyphenylacetic acid residues was bound to Choi, a structure not previously reported for other members of the aeruginosin family. We sequenced the complete genome of Nostoc sp. UHCC 0870 and identified the putative 36 kb varlaxin biosynthetic gene cluster. Bioinformatics analysis confirmed that varlaxins belong to the aeruginosin family of natural products. Varlaxins 1046A and 1022A strongly inhibited the three human trypsin isoenzymes with IC50 of 0.62-3.6 nM and 97-230 nM, respectively, including a prometastatic trypsin-3, which is a therapeutically relevant target in several types of cancer. These results substantially broaden the genetic and chemical diversity of the aeruginosin family and provide evidence that the aeruginosin family is a source of strong inhibitors of human serine proteases.

Plant-derived sulfur containing natural products produced as a response to biotic and abiotic stresses: A review of their structural diversity and medicinal importance

Plant-derived sulfur-containing secondary metabolites constitute a small group of low-molecular weight natural products, which play a vital role in plant-pest interactions in numerous plant families andrepresent major defense molecules in the Asteraceae, Alliaceae, and Brassicaceae families. In this review we highlight the crucial role of environmental stress factors in the production of S-containing secondary metabolites. Furthermore, we describe a serendipitous variety of plant-derived sulfur-containing natural products produced or induced under biotic and abiotic stress and their structural diversity, promising pharmacological properties for use by humans, and beneficial effects for plants. Specifically, cruciferous phytoalexins are known as elicit plant defense molecules. Glucosinolates are candidates for tumorpreventive effects. Cysteine sulfoxides found in garlic are considered as profound antimicrobial agents. In this review, we discuss types of S bonds in the molecules and their relevance for the medicinal effect as well as the biological activities of sulfur-containing secondary metabolites and possible future avenues.

Honokiol alleviates the degeneration of intervertebral disc via suppressing the activation of TXNIP-NLRP3 inflammasome signal pathway.

Intervertebral disc degeneration (IVDD) is a multifactorial disease and responsible for many spine related disorders, causes disability in the workforce and heavy social costs all over the world. Honokiol, a low molecular weight natural product, could penetrate into and distribute in IVDs to achieve therapeutic effect in a rat tail model. Therefore, the present study was undertaken to examine the antiinflammatory, antioxidation and IVD-protective effect of honokiol using nucleus pulposus cells and investigate its mechanisms to provide a new basis for future clinical treatment of IVDD. In the current study, we demonstrated that honokiol inhibits the H2O2-induced apoptosis (caspase-9, caspase-3, and bax), levels of oxidative stress mediators (ROS, MDA), expression of inflammatory mediators (Interleukin-6, COX-2, and iNOS), major matrix degrading proteases (MMP-3, MMP-13, ADAMTS5, and ADAMTS4) associated with nucleus pulposus degradation. Furthermore, we found nucleus pulposus protective ability of honokiol by up-regulating extra cellular matrix anabolic factors like type II collagen (Col II) and SOX9 in nucleus pulposus. We also found that honokiol suppressed the phosphorylation of NF-kB and JNK, and activation of TXNIP-NLRP3 inflammasome in H2O2-stimulated nucleus pulposus cells, thereby inhibiting the activation of downstream inflammatory mediators such as Interleukin-1β. Furthermore, honokiol showed a cartilage protective effect in the progression of IVDD in a rat model induced by puncture. Thus, our results demonstrate that honokiol inhibited the H2O2 induced apoptosis, oxidative stress, and inflammatory responses through the depression of TXNIP/NLRP3/caspase-1/ Interleukin - 1β signaling axis and the activation of NF-kB and JNK. Honokiol possess nucleus pulposus protective properties and may be of value in suppressing the pathogenesis of IVDD.

Metabolites of Trichoderma species isolated from damp building materials.

Buildings that have been flooded often have high concentrations of Trichoderma spores in the air while drying. Inhaled spores and spore and mycelial fragments contain large amounts of fungal glucan and natural products that contribute to the symptoms associated with indoor mould exposures. In this study, we considered both small molecules and peptaibol profiles of T. atroviride, T. koningiopsis, T. citrinoviride, and T. harzianum strains obtained from damp buildings in eastern Canada. Twenty-residue peptaibols and sorbicillin-derived metabolites (1-6) including a new structure, (R)-vertinolide (1), were characterized from T. citrinoviride. Trichoderma koningiopsis produced several koninginins (7-10), trikoningin KA V, and the 11-residue lipopeptaibols trikoningin KB I and trikoningin KB II. Trichoderma atroviride biosynthesized a mixture of 19-residue trichorzianine-like peptaibols, whereas T. harzianum produced 18-residue trichokindin-like peptaibols and the 11-residue harzianin HB I that was subsequently identified from the studied T. citrinoviride strain. Two α-pyrones, 6-pentyl-pyran-2-one (11) and an oxidized analog (12), were produced by both T. atroviride and T. harzianum. Aside from exposure to low molecular weight natural products, inhalation of Trichoderma spores and mycelial fragments may result in exposure to membrane-disrupting peptaibols. This investigation contributes to a more comprehensive understanding of the biologically active natural products produced by fungi commonly found in damp buildings.

Decidual Glycodelin-A regulates the phenotype and functions of human macrophages

Mycotoxins are low-molecular weight natural products produced as secondary metabolites by filamentous fungi and more than 500 mycotoxins with different physicochemical properties are currently known. Contamination by mycotoxins can result in liver and kidney diseases, nervous system damage, immunosuppression, and carcinogenicity. Consequently, the food safety authorities have set strict maximum permissible limits for mycotoxins. Over the past two decades numerous analytical techniques have been developed for mycotoxin detection in food. Recently, the application of nanotechnology in biosensors can range from transducer device to recognition ligand, label, and running systems. The application of nanomaterials in sensor development provides excellent advantages such as miniaturization of devices and signal enhancements. Such nanomaterial-based sensor devices can be sensitive, cost effective, and highly beneficial for the food industry ensuring on-site safety and preservation of food quality. This chapter reviews some recent developments on nanomaterial-based sensors developed for analysis of mycotoxin in food.

Synthetic Glycosides and Glycoconjugates of Low Molecular Weight Natural Products.

Enzymatically controlled transfer of saccharide moieties constitutes a fundamental biological process, essential for both primary and secondary metabolism. Natural products, including countless glycosides, with a rich tradition of use in ethnopharmacology, remain a prime source of inspiration for medicinal chemistry and molecular pharmacology, but their availability from biological sources is usually scarce, hampering attempts at application for new drug discovery and development. Chemical glycosylation on the other hand, although continuously undergoing sophisticated mechanistic studies, has nevertheless already matured as a set of methods which are able to provide substantial amounts of pure chemical entities: natural glycosides, as well as their congeners and mimics, necessary for the study of biological activity in quality assurance systems and required for drug development by pharmaceutical regulations. The paper presents a review of natural products and their analogues glycosylation, in a set of arbitrary selected examples, supplemented with comments on general advances in chemical glycosylation methodology and their applicability for particular categories of secondary metabolites.

Marine Low Molecular Weight Natural Products as Potential Cancer Preventive Compounds

Due to taxonomic positions and special living environments, marine organisms produce secondary metabolites that possess unique structures and biological activities. This review is devoted to recently isolated and/or earlier described marine compounds with potential or established cancer preventive activities, their biological sources, molecular mechanisms of their action, and their associations with human health and nutrition. The review covers literature published in 2003–2013 years and focuses on findings of the last 2 years.

Honokiol, a low molecular weight natural product, prevents inflammatory response and cartilage matrix degradation in human osteoarthritis chondrocytes

Proinflammatory cytokine interleukin-1β (IL-1β) stimulates several mediators of cartilage degradation and plays an important role in the pathogenesis of osteoarthritis (OA). Honokiol, a low molecular weight natural product isolated from the Magnolia officinalis, has been shown to possess anti-inflammatory effect. Here, we used an in vitro model of cartilage inflammation to investigate the therapeutic potential of honokiol in OA. Human OA chondrocytes were cultured and pretreated with honokiol (2.5-10 µM) with or without IL-1β (10 ng/ml). Nitric oxide (NO) production was quantified by Griess reagent. Prostaglandin (PG)E2 , metalloproteinase-13 (MMP-13), and interleukin-6 (IL-6) productions were quantified by enzyme-linked immunosorbent assay. The expressions of collagen II, cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), and nuclear factor κB (NF-κB)-related signaling molecules were determined by Western blotting. Our data showed that IL-1β markedly stimulated the expressions of iNOS and COX-2 and the productions of NO, PGE2 , and IL-6, which could be significantly reversed by honokiol. Honokiol could also suppress the IL-1β-triggered activation of IKK/IκBα/NF-κB signaling pathway. Moreover, honokiol significantly inhibited the IL-1β-induced MMP-13 production and collagen II reduction. Taken together, the present study suggests that honokiol may have a chondroprotective effect and may be a potential therapeutic choice in the treatment of OA patients.

Structural characterization of a flavonoid glycosyltransferase from Withania somnifera.

Medicinal plants are extensively utilized in traditional and herbal medicines, both in India and around the world due to the presence of diverse low molecular weight natural products such as flavonoids, alkaloids, terpenoids and sterols. Flavonoids which have health benefits for humans are the large class of phenylpropanoid-derived secondary metabolites and are mostly glycosylated by UDP-glycosyltransferases (UGTs). Although large numbers of different UGTs are known from higher plants, very few protein structures have been reported till now. In the present study, the three-dimensional model of flavonoid specific glycosyltransferases (WsFGT) from Withania somnifera was constructed based on the crystal structure of plant UGTs. The resulted model was assessed by various tools and the final refined model revealed GT-B type fold. Further, to understand the sugar donors and acceptors interactions with the active site of WsFGT, docking studies were performed. The amino acids from conserved PSPG box were interacted with sugar donor while His18, Asp110, Trp352 and Asn353 were important for catalytic function. This structural and docking information will be useful to understand the glycosylation mechanism of flavonoid glucosides.AbbreviationsDOPE - Discrete Optimized Potential Energy, PDB - Protein Data Bank, PSPG - Plant Secondary Product Glycosyltransferase, RMSD - Root Mean Squared Deviation, UDP - Uridine diphosphate, UGT - UDP-glycosyltransferases.

The inhibitory effect of honokiol, a natural plant product, on vestibular schwannoma cells

As the molecular biology of vestibular schwannoma (VS) is better understood, new means of targeting the pathways involved for intervention in schwannoma cells are being developed. Honokiol, a bioactive constituent of Magnolia officinalis, has attracted attention due to its diverse biological effects. This study was conducted to determine the inhibitory effect of honokiol on schwannoma cell proliferation. HEI 193 cells were used to investigate the growth-inhibitory effects of honokiol. Cell proliferation was assessed by MTT assays. Apoptosis was measured by flow cytometry analysis and immunofluorescence staining including Hoechst 33342 and TUNEL. Western blot analysis was used to assess the potential inhibition of extracellular signal-regulated kinase (ERK) and AKT signaling by honokiol. Honokiol exhibited significant antiproliferative activity in a dose-dependent manner on HEI 193 cells. Significant apoptosis was detected on schwannoma cells with 7 mg/mL(IC50) honokiol. Western blot analysis showed significant inhibition of ERK phosphorylation. Honokiol, a low molecular weight natural product, inhibits cell proliferation and promotes apoptosis in schwannoma cells by targeting the ERK pathway. Our data suggest that honokiol can be evaluated as a chemotherapeutic agent for VS.

Honokiol rescues sepsis-associated acute lung injury and lethality via the inhibition of oxidative stress and inflammation

Sepsis has a high mortality rate despite the recent advances in intensive care medicine and antibiotics. Honokiol, a low molecular weight natural product, is known to possess anti-inflammatory activity. Here, we investigate whether honokiol can ameliorate acute lung injury and lethal response in murine models of sepsis. Mice were intraperitoneally given vehicle or honokiol 30 min after the induction of sepsis by cecal ligation and puncture (CLP) and endotoxemia by administration of E. coli lipopolysaccharide (LPS). The productions of serum tumor necrosis factor-α (TNF-α), nitric oxide (NO), and high mobility group box 1 (HMGB 1) were increased in mice during sepsis, which could be reversed by honokiol. Honokiol could also effectively reduce the increased blood lipid peroxidation and nitrotyrosine in septic mice. Honokiol significantly reversed the inductions of inducible NO synthase and nuclear factor-κB (NF-κB) activation in the lungs of mice during sepsis. Honokiol also effectively rescued the lung edema, lung pathological changes, and lethality in septic mice. These findings suggest that honokiol is capable of suppressing the lethal response and acute lung injury associated with sepsis, and support the potential use of honokiol as a therapeutic agent for the conditions associated with septic shock.

4-Formylaminooxyvinylglycine, an Herbicidal Germination-Arrest Factor from Pseudomonas Rhizosphere Bacteria

A new oxyvinylglycine has been identified as a naturally occurring herbicide that irreversibly arrests germination of the seeds of grassy weeds, such as annual bluegrass (Poa annua), without significantly affecting the growth of established grass seedlings and mature plants or germination of the seeds of broadleaf plant species (dicots). Previously, Pseudomonas fluorescens WH6 and over 20 other rhizosphere bacteria were isolated and selected for their ability to arrest germination of P. annua seeds. The germination-arrest factor (GAF, 1) responsible for this developmentally specific herbicidal action has now been isolated from the culture filtrate of P. fluorescens WH6. Purification of this highly polar, low molecular weight natural product allowed its structure to be assigned as 4-formylaminooxy-l-vinylglycine on the basis of NMR spectroscopic and mass spectrometric data, in combination with D/L-amino acid oxidase reactions to establish the absolute configuration. Assay results for P. annua inhibition by related compounds known to regulate plant growth are presented, and a cellular target for 1 is proposed. Furthermore, using bioassays, TLC, and capillary NMR spectroscopy, it has been shown that GAF (1) is secreted by all other herbicidally active rhizosphere bacteria in our collection.

Cross‐linked agarose for separation of low molecular weight natural products in hydrophilic interaction liquid chromatography

Following its market introduction in 1982, the cross-linked 12% agarose gel media Superose 12 has become widely known as a tool for size exclusion chromatography of proteins and other biological macromolecules. In this review it is shown that, when appropriate mobile phases are used, Superose possesses adsorption properties similar to that of traditional media for hydrophilic interaction liquid chromatography (HILIC). This is illustrated by the separation and purification of low molecular weight compounds such as polyphenols including active components of traditional Chinese medicinal herbs and green tea. Structural features of the cross-linked agarose that likely cause the observed adsorption effects are discussed as well. These are identified as being primarily ether bonds acting as strong hydrogen bond acceptors as well as hydrophobic residues originating from the cross-linking reagents.

Characterization of cellular accumulation and toxicity of illudin S in sensitive and nonsensitive tumor cells.

Illudins are novel low molecular weight natural products cytotoxic to human tumor cells in vitro. Illudin-derived analogs are effective against experimental human cancers nonresponsive to conventional anticancer agents. It is not known why some illudin analogs are more efficacious in vitro and in vivo than other analogs. Therefore, the in vitro cytotoxicity of the parent compound illudin S towards tumor cells was characterized using radiolabeled drug. Two cell lines sensitive at nanomolar concentrations using only a 15-min exposure period displayed a saturable, energy-dependent accumulation of illudins with relatively low K(m) and high Vmax values. A nonsensitive cell line, requiring millimolar concentrations to achieve in vitro toxicity, showed minimal illudin uptake with higher K(m) and lower Vmax values. No release of radioactivity could be demonstrated from tumor cells, indicating that there was no efflux of illudin S (or metabolites) from these cells. The number of intracellular illudin S molecules required to kill 50% of cells of different tumor cell lines varied from 78000 to 1114000 molecules per cell and was correlated with the 2-h IC50 value determined using a colony-forming assay. Illudin S was cytotoxic to a variety of multidrug-resistant tumor cell lines regardless of whether resistance was mediated by gp170/mdrl, gp180/MRP, GSHTR-pi, topoisomerase I, topoisomerase II, increased DNA repair capacity, or alterations in intracellular thiol content. Information obtained in this study could be used to design clinical phase I trials and to develop analogs with improved therapeutic indexes.

From molecular genetics and secondary metabolism to molecular metabolites and secondary genetics

Secondary metabolites constitute a huge array of low molecular weight natural products that cannot be easily defined. Largely produced by bacteria, fungi, and green plants, they tend to be synthesized after active growth has ceased, in families of similar compounds, often at the same time as species-specific morphological characters become apparent. Although, in many cases, the function that the secondary metabolite performs in the producing organism is unknown, the bioactivity of these compounds has been exploited since prehistoric times as drugs, poisons, food flavoring agents, and so forth. In fungi, the polyketide family is the largest known group of secondary metabolite compounds. Polyketides are synthesized from acetate by a mechanism analogous to fatty acid biosynthesis but involving changes in oxidation level and stereochemistry during the chain-elongation process. The fungal polyketide biosynthetic pathways for aflatoxin and patulin have emerged as model systems. The use of blocked mutants has been an essential part of the research approach for both pathways. Molecular methods of studying fungal secondary metabolites were first used with penicillin and cephalosporin, both of which are amino acid derived. Most of the basic molecular work on polyketides was done with streptomycete-derived compounds; however, enough fungal data are now available to compare fungal and streptomycete polyketide synthases, as well as to map the genes involved in a number of polyketide pathways from both groups. The traditional dogma, derived from classical genetics, that genes for fungal pathways are unlinked, has been overturned. In addition, cloning of structural genes facilitates the formation of hybrid molecules, and we are on the brink of understanding certain regulatory functions. Key words: fungal metabolism, secondary metabolism, polyketide, β-lactam, product discovery.