A Homolog of the Histidine Kinase RetS Controls the Synthesis of Alginates, PHB, Alkylresorcinols, and Motility in Azotobacter vinelandii.

The two-component system GacS/A and the posttranscriptional control system Rsm constitute a genetic regulation pathway in Gammaproteobacteria; in some species of Pseudomonas, this pathway is part of a multikinase network (MKN) that regulates the activity of the Rsm system. In this network, the activity of GacS is controlled by other kinases. One of the most studied MKNs is the MKN-GacS of Pseudomonas aeruginosa, where GacS is controlled by the kinases RetS and LadS; RetS decreases the kinase activity of GacS, whereas LadS stimulates the activity of the central kinase GacS. Outside of the Pseudomonas genus, the network has been studied only in Azotobacter vinelandii. In this work, we report the study of the RetS kinase of A. vinelandii; as expected, the phenotypes affected in gacS mutants, such as production of alginates, polyhydroxybutyrate, and alkylresorcinols and swimming motility, were also affected in retS mutants. Interestingly, our data indicated that RetS in A. vinelandii acts as a positive regulator of GacA activity. Consistent with this finding, mutation in retS also negatively affected the expression of small regulatory RNAs belonging to the Rsm family. We also confirmed the interaction of RetS with GacS, as well as with the phosphotransfer protein HptB.

Deciphering the biotransformation mechanism of dialkylresorcinols by CYP4F11.

Cytochrome P450 enzymes (CYPs) are one of the most important classes of oxidative enzymes in the human body, carrying out metabolism of various exogenous and endogenous substrates. In order to expand the knowledge of these enzymes' specificity and to obtain new natural product derivatives, CYP4F11, a cytochrome P450 monooxygenase, was used in the biotransformation of dialkylresorcinols 1 and 2, a pair of antibiotic microbial natural products. This investigation resulted in four biotransformation products including two oxidative products: a hydroxylated derivative (3) and a carboxylic acid derivative (4). In addition, acetylated (5) and esterified products (6) were isolated, formed by further metabolism by endogenous yeast enzymes. Oxidative transformations were highly regioselective, and took place exclusively at the ω-position of the C-5 alkyl chain. Homology modeling studies revealed that optimal hydrogen bonding between 2 and the enzyme can only be established with the C-5 alkyl chain pointing towards the heme. The closely-related CYP4F12 was not capable of oxidizing the dialkylresorcinol 2. Modeling experiments rationalize these differences by the different shapes of the binding pockets with respect to the non-oxidized alkyl chain. Antimicrobial testing indicated that the presence of polar groups on the side-chains reduces the antibiotic activity of the dialkylresorcinols.

Discovery of anti-infective adipostatins through bioactivity-guided isolation and heterologous expression of a type III polyketide synthase.

Antibiotic resistance and emerging viral pandemics have posed an urgent need for new anti-infective drugs. By screening our microbial extract library against the main protease of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the notorious ESKAPE pathogens, an active fraction was identified and purified, leading to an initial isolation of adipostatins A (1) and B (2). In order to diversify the chemical structures of adipostatins toward enhanced biological activities, a type III polyketide synthase was identified from the native producer, Streptomyces davawensis DSM101723, and was subsequently expressed in an E. coli host, resulting in the isolation of nine additional adipostatins 3-11, including two new analogs (9 and 11). The structures of 1-11 were established by HRMS, NMR, and chemical derivatization, including using a microgram-scale meta-chloroperoxybenzoic acid epoxidation-MS/MS analysis to unambiguously determine the double bond position in the alkyl chain. The present study discovered SARS-CoV-2 main protease inhibitory activity for the class of adipostatins for the first time. Several of the adipostatins isolated also exhibited antimicrobial activity against selected ESKAPE pathogens.

Weak Interactions of the Isomers of Phototrexate and Two Cavitand Derivatives.

The interactions of two conformers of newly synthesized photoswitchable azobenzene analogue of methotrexate, called Phototrexate, with two cavitand derivatives, have been investigated in dimethyl sulfoxide medium. Photoluminescence methods have been applied to determine the complex stabilities and the related enthalpy and entropy changes associated to the complex formation around room temperature. Results show opposite temperature dependence of complex stabilities. The structure of the upper rims of the host molecules and the reordered solvent structure were identified as the background of the opposite tendencies of temperature dependence at molecular level. These results can support the therapeutic application of the photoswitchable phototrexate, because the formation of inclusion complexes is a promising method to regulate the pharmacokinetics of drug molecules.

Dual-Readout Tyrosinase Activity Assay Facilitated by a Chromo-Fluorogenic Reaction between Catechols and Naphthoresorcin.

Analyte-responsive chromo-fluorogenic reactions under accessible conditions are important for designing small-molecule spectroscopic probes. We describe a series of newly constructed motifs based on the chromo-fluorogenic reaction between catechol derivatives (typically hydroxytyrosol, dopamine, and levodopa) and naphthoresorcin (NR) in aqueous solution under ambient conditions. The weakly absorptive and fluorogenic catechols/NR was converted to products having visible absorption and bright fluorescence within several minutes. The chromo-fluorophores produced from this reaction had a maximum absorbance at 458 nm and emission at 480 nm with high fluorescence quantum yields (30-84%). Inspired by the tyrosinase-catalyzed hydroxylation of monophenols to catechols, the tyrosinase-enabled chromo-fluorogenic reaction was verified by using monophenol (typically tyrosol) as the substrate. In this regard, a dual-readout tyrosinase activity assay was developed by virtue of the in situ "turn-on" optical signals. Furthermore, a test of tyrosinase inhibition, by using a common inhibitor kojic acid, demonstrated the potential of the chromo-fluorogenic reaction for developing other tyrosinase related assays and signal transduction.

Study of the sRNA RsmY involved in the genetic regulation of the synthesis of alginate and alkyl resorcinols in Azotobacter vinelandii.

Azotobacter vineladii is a Gram-negative bacterium that produces alginate and poly-hydroxybutyrate (PHB), two polymers of biotechnological interest. This bacterium has the ability to form desiccation-resistant cysts. In the cyst the membrane phospholipids are replaced with a family of phenolic lipids called alkylresorcinols (ARs). The alginate, PHB, and ARs are controlled by the GacS/A two-component system and the small regulatory RNA (sRNA) RsmZ1, belonging to the Rsm (Csr) regulatory system. The Rsm (Csr) systems usually possess two or more sRNAs, in this regard A. vinelandii is the bacterium with the highest number of rsm-sRNAs. Originally, the presence of two sRNAs of the RsmY family (RsmY1 and RsmY2) was reported, but in a subsequent work it was suggested that they conformed to a single sRNA. In this work we provide genetic evidence confirming that rsmY1 and rsmY2 constitute a single gene. Also, it was established that rsmY mutation decreased alginate and ARs production, but did not affect the PHB synthesis. Transcriptional studies showed that rsmY has its higher expression during the stationary growth phase, and in the absence of RsmZ1, rsmY increases its transcription. Interestingly, rsmY expression was influenced by the carbon source, but its expression did not correlate with alginate production.

Isolation of three new meroterpenoids and seven known compounds from Albatrellus yasudae and their Aß-aggregation inhibitory activity.

Alzheimer's disease is a serious neurologic disorder that cannot be cured completely. In this study, we targeted compounds that inhibit amyloid-beta (Aß) aggregation, based on the amyloid cascade hypothesis. Ten compounds (1-10) were isolated from CHCl3 extracts of the mushroom Albatrellus yasudae using Aß-aggregation inhibitory activity-guided separation. The structures of these compounds were elucidated from 1D and 2D NMR and MS spectral data. Compounds 1-3 were novel, whereas 4-10 were identified as the known compounds grifolin, grifolic acid, neogrifolin, confluentin, 2-hydroxyneogrifolin, daurichromenic acid, and a cerebroside derivative. Compounds 1-10 were tested for Aß-aggregation inhibitory activity. Compounds 1, 3, 5, 6, 8, and 9 have potential as Aß-aggregation inhibitory activity.

Comparison of the metabolism of 10 cosmetics-relevant chemicals in EpiSkin™ S9 subcellular fractions and in vitro human skin explants.

An understanding of the bioavailability of topically applied cosmetics ingredients is key to predicting their local skin and systemic toxicity and making a safety assessment. We investigated whether short-term incubations with S9 from the reconstructed epidermal skin model, EpiSkin™, would give an indication of the rate of chemical metabolism and produce similar metabolites to those formed in incubations with human skin explants. Both have advantages: EpiSkin™ S9 is a higher-throughput assay, while the human skin explant model represents a longer incubation duration (24 hours) model integrating cutaneous distribution with metabolite formation. Here, we compared the metabolism of 10 chemicals (caffeine, vanillin, cinnamyl alcohol, propylparaben, 4-amino-3-nitrophenol, resorcinol, 4-chloroaniline, 2-amino-3-methyl-3H-imidazo[4,5-F]quinoline and 2-acetyl aminofluorene) in both models. Both models were shown to have functional Phase 1 and 2 enzymes, including cytochrome P450 activities. There was a good concordance between the models with respect to the level of metabolism (stable vs. slowly vs. extensively metabolized chemicals) and major early metabolites produced for eight chemicals. Discordant results for two chemicals were attributed to a lack of the appropriate cofactor (NADP+ ) in S9 incubations (cinnamyl alcohol) and protein binding influencing chemical uptake in skin explants (4-chloroaniline). These data support the use of EpiSkin™ S9 as a screening assay to provide an initial indication of the metabolic stability of a chemical applied topically. If required, chemicals that are not metabolized by EpiSkin™ S9 can be tested in longer-term incubations with in vitro human explant skin to determine whether it is slowly metabolized or not metabolized at all.

Bacterial Autoimmune Drug Metabolism Transforms an Immunomodulator into Structurally and Functionally Divergent Antibiotics.

Tapinarof is a stilbene drug that is used to treat psoriasis and atopic dermatitis, and is thought to function through regulation of the AhR and Nrf2 signaling pathways, which have also been linked to inflammatory bowel diseases. It is produced by the gammaproteobacterial Photorhabdus genus, which thus represents a model to probe tapinarof structural and functional transformations. We show that Photorhabdus transforms tapinarof into novel drug metabolism products that kill inflammatory bacteria, and that a cupin enzyme contributes to the conversion of tapinarof and related dietary stilbenes into novel dimers. One dimer has activity against methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecalis (VRE), and another undergoes spontaneous cyclizations to a cyclopropane-bridge-containing hexacyclic framework that exhibits activity against Mycobacterium. These dimers lack efficacy in a colitis mouse model, whereas the monomer reduces disease symptoms.

Chemistry, bioactivity and biosynthesis of cyanobacterial alkylresorcinols.

Covering: up to 2019 Alkylresorcinols are amphiphilic metabolites, well-known for their diverse biological activities, produced by both prokaryotes and eukaryotes. A few classes of alkylresorcinol scaffolds have been reported from the photoautotrophic cyanobacteria, ranging from the relatively simple hierridins to the more intricate cylindrocyclophanes. Recently, it has emerged that cyanobacteria employ two different biosynthetic pathways to produce unique alkylresorcinol scaffolds. However, these convergent pathways intersect by sharing biosynthetic elements which lead to common structural motifs. To obtain a broader view of the biochemical diversity of these compounds in cyanobacteria, we comprehensively cover the isolation, structure, biological activity and biosynthesis of their mono- and dialkylresorcinols. Moreover, we provide an overview of the diversity and distribution of alkylresorcinol-generating biosynthetic gene clusters in this phylum and highlight opportunities for discovery of novel alkylresorcinol scaffolds. Because some of these molecules have inspired notable syntheses, different approaches used to build these molecules in the laboratory are showcased.

DbdR, a New Member of the LysR Family of Transcriptional Regulators, Coordinately Controls Four Promoters in the Thauera aromatica AR-1 3,5-Dihydroxybenzoate Anaerobic Degradation Pathway.

The facultative anaerobe Thauera aromatica strain AR-1 uses 3,5-dihydroxybenzoate (3,5-DHB) as a sole carbon and energy source under anoxic conditions using an unusual oxidative strategy to overcome aromatic ring stability. A 25-kb gene cluster organized in four main operons encodes the anaerobic degradation pathway for this aromatic. The dbdR gene coding for a LysR-type transcriptional regulator (LTTR), which is present at the foremost end of the cluster, is required for anaerobic growth on 3,5-DHB and for the expression of the main pathway operons. A model structure of DbdR showed conserved key residues for effector binding with its closest relative TsaR for p-toluenesulfonate degradation. We found that DbdR controlled expression of three promoters upstream from the operons coding for the three main steps of the pathway. While one of them (P orf20 ) was only active in the presence of 3,5-DHB, the other two (P dbhL and P orf18 ) showed moderate basal levels that were further induced in the presence of the pathway substrate, which needed be converted to hydroxyhydroquinone to activate transcription. Both basal and induced activities were strictly dependent on DbdR, which was also required for transcription from its own promoter. DbdR basal expression was moderately high and, unlike most LTTR, increased 2-fold in response to the presence of the effector. DbdR was found to be a tetramer in solution, producing a single retardation complex in binding assays with the three enzymatic promoters, consistent with its tetrameric structure. The three promoters had a conserved organization with a clear putative primary (regulatory) binding site and a putative secondary (activating) binding site positioned at the expected distances from the transcription start site. In contrast, two protein-DNA complexes were observed for the P dbdR promoter, which also showed significant sequence divergence from those of the three other promoters. Taken together, our results show that a single LTTR coordinately controls expression of the entire 3,5-DHB anaerobic degradation pathway in Thauera aromatica AR-1, allowing a fast and optimized response to the presence of the aromatic.IMPORTANCEThauera aromatica AR-1 is a facultative anaerobe that is able to use 3,5-dihydroxybenzoat (3,5-DHB) as the sole carbon and energy source in a process that is dependent on nitrate respiration. We have shown that a single LysR-type regulator with unusual properties, DbdR, controls the expression of the pathway in response to the presence of the substrate; unlike other regulators of the family, DbdR does not repress but activates its own synthesis and is able to bind and activate three promoters directing the synthesis of the pathway enzymes. The promoter architecture is conserved among the three promoters but deviates from that of typical LTTR-dependent promoters. The substrate must be metabolized to an intermediate compound to activate transcription, which requires basal enzyme levels to always be present. The regulatory network present in this strain is designed to allow basal expression of the enzymatic machinery, which would rapidly metabolize the substrate when exposed to it, thus rendering the effector molecule. Once activated, the regulator induces the synthesis of the entire pathway through a positive feedback, increasing expression from all the target promoters to allow maximum growth.

Structure of Hierridin C, Synthesis of Hierridins B and C, and Evidence for Prevalent Alkylresorcinol Biosynthesis in Picocyanobacteria.

Small, single-celled planktonic cyanobacteria are ubiquitous in the world's oceans yet tend not to be perceived as secondary metabolite-rich organisms. Here we report the isolation and structure elucidation of hierridin C, a minor metabolite obtained from the cultured picocyanobacterium Cyanobium sp. LEGE 06113. We describe a simple, straightforward synthetic route to the scarcely produced hierridins that relies on a key regioselective halogenation step. In addition, we show that these compounds originate from a type III PKS pathway and that similar biosynthetic gene clusters are found in a variety of bacterial genomes, most notably those of the globally distributed picocyanobacteria genera Prochlorococcus, Cyanobium and Synechococcus.

4-Methyl-5-Pentylbenzene-1,3-Diol Regulates Chemotactic Cell Aggregation and Spore Maturation Via Different Mechanisms in Dictyostelium discoideum.

4-Methyl-5-pentylbenzene-1,3-diol (MPBD), a product of the polyketide synthase SteelyA, is a signaling molecule that regulates Dictyostelium discoideum development. During early development, MPBD controls chemotactic cell aggregation by regulating the expression of genes in the cAMP signaling pathway; however, during culmination at late development, it induces spore maturation. In the present study, we analyzed the effects of MPBD, its derivatives, and a putative MPBD-derived metabolite on developmental defects in the MPBD-less stlA null mutant. Using structure-activity relationship studies, it was observed that in MPBD, the functional groups that were essential for induction of spore maturation were different from those essential for induction of cell aggregation. Dictyoquinone, a putative MPBD metabolite rescued the aggregation defect in stlA null mutant in early development, but not the spore maturation defect at the later stage. Our data suggest that MPBD regulates chemotactic cell aggregation and spore maturation via different mechanisms.

Antispasmodic Activity of Prenylated Phenolic Compounds from the Root Bark of Morus nigra.

Black mulberry is a widely acknowledged ancient traditional medicine. Its extract and constituents have been reported to exert various bioactivities including antimicrobial, hypotensive, analgesic etc. effects. While black mulberry preparations are also used as antispasmodic agents in folk medicine, no related studies are available on its isolated constituents. Through an extensive chromatographic purification, seven phenolic compounds were isolated from the methanol extract of Morus nigra root bark, including morusin (1), kuwanon U (2), kuwanon E (3), moracin P (4), moracin O (5), albanol A (6), and albanol B (7). A complete NMR signal assignment of moracin P and O was achieved, and related literature errors confusing the identity of moracin derivatives are hereby clarified. Compounds 2, 5 and 7 were identified as strong antispasmodic agents on isolated rat ileum and tracheal smooth muscles, while compound 3, a methoxy derivative of 2, was inactive. Moracin O (5) inhibited the ileal and tracheal smooth muscle contractions with Emax values of 85% and 302 mg, respectively. Those actions were superior as compared with papaverine. Our findings demonstrate that prenylated arylbenzofurans, geranylated flavonoids and Diels-Alder adducts from Morus nigra are valuable antispasmodic agents. Compounds 2, 5 and 7 are suggested as marker compounds for quality control of antispasmodic mulberry preparations. Moracin O (5) is a new lead compound for related drug development initiatives.

Targeted Metabolite Profiling-Based Identification of Antifungal 5-n-Alkylresorcinols Occurring in Different Cereals against Fusarium oxysporum.

A rapid and convenient biochemometrics-based analysis of several cereal-derived extracts was used to identify n-alkyl(enyl)resorcinols (AR) as antifungals against Fusarium oxysporum. Total AR content and liquid chromatography/mass spectrometry (LC-MS)-based profiles were recorded for each extract, in addition to their antifungal activity, to help integrate these chemical and biological datasets by orthogonal partial least squares regression. In this study, we developed and used a micro-scale amended medium (MSAM) assay to evaluate the in vitro mycelial growth inhibition at low amounts of extracts. Triticale husk-derived extracts had the highest AR content (662.1 µg olivetol equivalent/g dry extract), exhibiting >79% inhibition at the highest doses (10.0⁻1.0 µg/µL). Correlation of the chemical and antifungal datasets using supervised metabolite profiling revealed that 5-n-nonadecanylresorcinol, 5-n-heneicosylresorcinol, and 5-n-tricosyl-resorcinol were the most active ARs occurring in cereal products from Colombia. Hence, we propose the biochemometrics-based approach as a useful tool for identifying AR-like antifungals against F. oxysporum.

Antidiabetic potential of phytochemicals isolated from the stem bark of Myristica fatua Houtt. var. magnifica (Bedd.) Sinclair.

Phytochemical investigation of the stem bark of Myristica fatua Houtt. led to the isolation of a new compound 1 (3-tridecanoylbenzoic acid), along with six known acylphenols (2-7). All the compounds displayed moderate inhibitory activity on α-amylase and significant activity on α-glucosidase; however malabaricone B (6) and C (7) were identified as potent α-glucosidase inhibitors with IC50 values of 63.70 ±â€¯0.546, and 43.61 ±â€¯0.620 µM respectively. Acylphenols (compounds 3-7) also showed significant antiglycation property. The molecular docking and dynamics simulation studies confirmed the efficient binding of malabaricone C with C-terminus of human maltase-glucoamylase (2QMJ). Malabaricone B also enhanced the 2-NBDG [2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxy glucose] uptake in L6 myotubes. These findings demonstrate that acylphenols isolated from Myristica fatua Houtt. can be considered as a lead scaffold for the treatment of type II diabetes mellitus.

Dialkylresorcinols as bacterial signaling molecules.

It is well recognized that bacteria communicate via small diffusible molecules, a process termed quorum sensing. The best understood quorum sensing systems are those that use acylated homoserine lactones (AHLs) for communication. The prototype of those systems consists of a LuxI-like AHL synthase and a cognate LuxR receptor that detects the signal. However, many proteobacteria possess LuxR receptors, yet lack any LuxI-type synthase, and thus these receptors are referred to as LuxR orphans or solos. In addition to the well-known AHLs, little is known about the signaling molecules that are sensed by LuxR solos. Here, we describe a novel cell-cell communication system in the insect and human pathogen Photorhabdus asymbiotica. We identified the LuxR homolog PauR to sense dialkylresorcinols (DARs) and cyclohexanediones (CHDs) instead of AHLs as signals. The DarABC synthesis pathway produces the molecules, and the entire system emerged as important for virulence. Moreover, we have analyzed more than 90 different Photorhabdus strains by HPLC/MS and showed that these DARs and CHDs are specific to the human pathogen P. asymbiotica. On the basis of genomic evidence, 116 other bacterial species are putative DAR producers, among them many human pathogens. Therefore, we discuss the possibility of DARs as novel and widespread bacterial signaling molecules and show that bacterial cell-cell communication goes far beyond AHL signaling in nature.

Recognition and sequestration of ω-fatty acids by a cavitand receptor.

One of the largest driving forces for molecular association in aqueous solution is the hydrophobic effect, and many synthetic receptors with hydrophobic interiors have been devised for molecular recognition studies in water. Attempts to create the longer, narrower cavities appropriate for long-chain fatty acids have been thwarted by solvophobic collapse of the synthetic receptors, giving structures that have no internal spaces. The collapse generally involves the stacking of aromatic panels onto themselves. We describe here the synthesis and application of a deep cavitand receptor featuring "prestacked" aromatic panels at the upper rim of the binding pocket. The cavitand remains open and readily sequesters biologically relevant long-chain molecules-unsaturated ω-3, -6, and -9 fatty acids and derivatives such as anandamide-from aqueous media. The cavitand exists in isomeric forms with different stacking geometries and n-alkanes were used to characterize the binding modes and conformational properties. Long alkyl chains are accommodated in inverted J-shaped conformations. An analogous cavitand with electron-rich aromatic walls was prepared and comparative binding experiments indicated the role of intramolecular stacking in the binding properties of these deep container molecules.

Application of hydrolases and probiotic Pediococcus acidilactici BaltBio01 strain for cereal by-products conversion to bioproduct for food/feed.

The aim of this study was to apply the enzymatic treatment and fermentation by Pediococcus acidilactici BaltBio01 strain for industrial cereal by-products conversion to food/feed bioproducts with high amount of probiotic lactic acid bacteria (LAB). LAB propagated in potato media and spray-dried remained viable during 12 months (7.0 log10 cfu/g) of storage and was used as a starter for cereal by-products fermentation. The changes of microbial profile, biogenic amines (BAs), mycotoxins, lactic acid (L+/D-), lignans and alkylresorcinols (ARs) contents in fermented cereal by-product were analysed. Cereal by-products enzymatic hydrolysis before fermentation allows to obtain a higher count of LAB during fermentation. Fermentation with P. acidilactici reduce mycotoxins content in fermented cereal by-products. According to our results, P. acidilactici multiplied in potato juice could be used for cereal by-products fermentation, as a potential source to produce safer food/feed bioproduct with high amount of probiotic LAB for industrial production.

Delivery and Release of Small-Molecule Probes in Mitochondria Using Traceless Linkers.

Mitochondria-penetrating peptides (MPPs) are specific targeting vectors for the localization of small molecules to the mitochondrial matrix. Mitochondrial targeting of small molecules has enabled the development of a number of potential therapeutics and chemical probes. However, the need for covalent conjugation of small molecules to MPPs can negatively affect the activity of the appended cargo against its cellular target. Here, we describe cleavable linkers designed for the traceless release of chemical cargo from MPPs following mitochondrial transit. The cleavage kinetics of a number of disulfides were investigated using a fluorescent reporter system in order to optimize linker stability for mitochondrial release. The stability of mono- and disubstituted disulfides was determined to be sufficient during transit through the cytosol while still allowing for release of the cargo within 24 h. This linker system successfully released the compound Luminespib, an HSP90 inhibitor, which was deactivated by direct MPP conjugation. The releasable conjugate regenerated Luminespib activity and induced mitochondrial phenotypes of HSP90 inhibition. This linker may prove useful in expanding the repertoire of small molecules that can be used with mitochondrial targeting vectors.