Frontiers in Chemistry

N-methyl-D-aspartate receptors (NMDARs) are crucial therapeutic targets, modulated by endogenous neurosteroids like pregnenolone sulfate (PES). This study investigates a novel structure-activity relationship approach focusing on the steroidal D-ring, employing the bioisosteric replacement of C-17 or C-20 keto groups with oximes and oxime ethers. We synthesized a series of pregn-5-ene and androst-5-ene derivatives (11-23) and evaluated their positive allosteric modulator (PAM) activity on recombinant rat GluN1/GluN2B receptors via patch-clamp in HEK293 cells. Our study revealed that pregnenolone-derived C-20 oxime ethers are potent and efficacious PAMs of NMDAR. Several analogues have been demonstrated as more potent than PES (Emax = 116%; EC50 = 21.7 {micro}M). Compound 12 (C-20 ethyl oxime ether, C-3 hemiglutarate) displayed the highest efficacy, potentiating NMDAR currents over 6-fold more than PES (Emax = 673 {+/-} 121%; EC50 = 8.7 {+/-} 1.1 {micro}M). Compound 17 (C-20 methyl oxime ether analogue) exhibited the highest potency, being over 3.5-fold more potent than PES (Emax = 503 {+/-} 68%; EC50 = 6.1 {+/-} 0.4 {micro}M). In contrast, some C-17 analogues and derivatives with bulkier C-20 oxime substituents showed complex modulatory behavior. Promisingly, key compounds demonstrated favorable in vitro ADME profiles, including high metabolic stability and, for 12, excellent thermodynamic solubility. These results validate C-20 oxime ether modification of the pregnenolone scaffold as an effective strategy for generating potent NMDAR PAMs with potentially superior efficacy and drug-like properties compared to endogenous modulators.

The lack of nuclear estrogen receptor (ER and ER{beta}) bindings of 10{beta}-hydroxyestra-1,4-diene-3,17-dione (HEDD) and structurally related steroidal para-quinols have been shown by an extensive series of multidisciplinary investigational evidence including specific receptor binding studies. In support of the latter, the absence of estrogen-derived para-quinols in vivo uterotrophic effects has also been well documented. Via in silico docking, a recent publication by Canario et al. (2022) reported a robust binding of HEDD (Figure 1B) to ER. The authors claimed a strong binding of HEDD -- as strong as that of its natural ligand, 17{beta}-estradiol (E2), the main human estrogen. However, an examination of the virtual binding pocket revealed that at least one residue near the critical ligand-binding site of their reported HEDD-ER complex was labelled as "unknown" indicating thereby alteration of the receptors published structure (Tannenbaum et al, 1998; Bafna et al., 2020) to fit the ligand. Based on these arguments, the contradictory result by Canario et al. (2022) on HEDDs binding to ER should be dismissed. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=102 SRC="FIGDIR/small/501604v2_fig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@488472org.highwire.dtl.DTLVardef@ef7642org.highwire.dtl.DTLVardef@13d1ff4org.highwire.dtl.DTLVardef@1fce259_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 1.C_FLOATNO (A) Schematic illustration of CNS-selective reductive bioactivation of bioprecursor prodrugs shown in panel B to the corresponding estrogen (E2, E2 or E1). (B) Chemical structures of bioprecursor prodrugs of estrogens: 10,17-dihydroxyestra-1,4-dien-3-one (DHED) for E2; 10,17-dihydroxyestra-1,4-dien-3-one (DHED) for E2, and 10-hydroxyestra-1,4-dien-3,17-dione (HEDD) for E1 (Prokai-Tatrai and Prokai, 2018). C_FIG

Soluble epoxide hydrolase (sEH) is a bifunctional enzyme responsible for lipid metabolism and is a promising drug target. Here, we report the first-in-class PROTACs small molecule degraders of sEH. Our optimized PROTAC selectively targets the degradation of cytosolic but not peroxisomal sEH, resulting in exquisite spatiotemporal control. Remarkably, our sEH PROTAC molecule has higher potency in cellular assays compared to the parent sEH inhibitor as measured by significantly reduced ER stress. Interestingly, our mechanistic data indicate that our PROTAC directs degradation of cytosolic sEH via the lysosome, not through the proteasome. The molecules presented here are useful chemical probes to study the biology of sEH with the potential for therapeutic development. Broadly, our results represent a proof-of-concept for the superior cellular potency of sEH degradation over sEH enzymatic inhibition, as well as subcellular compartment-selective modulation of a protein by PROTACs. HighlightsO_LIFirst-in-class soluble epoxide hydrolase (sEH) small-molecule degraders. C_LIO_LISelective degradation of cytosolic but not peroxisomal sEH. C_LIO_LISignificant and stable reduction in sEH protein levels, leading to enhanced cellular efficacy in ER stress reduction relative to the parent inhibitor. C_LI

Background and purposeNeurosteroids modulate neuronal function and are promising therapeutic agents for neuropsychiatric disorders. Neurosteroid analogues are approved for treating postpartum depression and are of interest in other disorders. GABA-A receptors are well characterized targets of natural neurosteroids, but other biological pathways are likely relevant to therapeutic mechanisms and/or to off-target effects. We performed hypothesis-generating in silico analyses and broad in vitro biological screens to assess the range of actions of neurosteroids analogues of varying structural attributes. Key ResultsWe employed in silico molecular similarity analysis and network pharmacology to elucidate likely targets. This analysis confirmed likely targets beyond GABA-A receptors. We then functionally screened 19 distinct neurosteroid structures across 78 targets representing interconnected signaling pathways, complemented with a limited screen of kinase activation. Results revealed unanticipated modulation of targets by neurosteroids with some structural selectivity. Many compounds-initiated androgen receptor translocation with little or no enantioselectivity. Modulation of multiple G-protein receptors was also unexpected. Conclusions and implicationsNeurosteroids are ascendant treatments in neuropsychiatry, but their full spectrum of actions remains unclear. This virtual and biological screening discovery approach opens new vistas for exploring mechanism of neurosteroids analogues. The multifaceted approach provides an unbiased, holistic exploration of the potential effects of neurosteroids across various molecular targets and provides a platform for future validation studies to aid drug discovery.

Insecticide resistance in mosquitoes, largely mediated by cytochrome P450 monooxygenases (CYPs), compromises the efficacy of vector control tools. In this study, chemically-wise selected natural extracts and compounds were screened for their CYP inhibition potential. Among 37 tested plant extracts and fractions, a decarboxylated acidic fraction of industrial hemp (Cannabis sativa Linnaeus var. Futura 75) emerged as a promising hit, and phytochemical profiling identified cannabidiol (CBD) as its major component (IC = 18.37 M for CYP9K1). CBD was used as a scaffold to generate semisynthetic analogues; of which a piperazinyl analogue outperformed the natural scaffold demonstrating significantly greater potency (IC = 2.50 M for CYP9K1). Docking studies using homology-derived CYP9K1 models also supported a stronger binding affinity of the piperazinyl analogue relative to CBD. Toxicity assays using pyrethroid-resistant Anopheles gambiae Giles adults confirmed that neither CBD nor the piperazinyl analogue had intrinsic toxicity, yet the semisynthetic analogue significantly enhanced deltamethrin efficacy, showing a threefold synergistic effect. The safety profile of the cannabis compounds for non-target organisms was evaluated through human cell line cytotoxicity tests and bee toxicity assays, suggesting low non-target organism toxicity. Our study describes the identification of a plant-derived synergist lead with strong potential as an insecticide additive to combat metabolic resistance in malaria-transmitting mosquitoes.

Collections of natural extracts hold potential for the discovery of novel natural products with original modes of action. The prioritization of extracts from collections remains challenging due to the lack of workflow that combines multiple-source information to facilitate the data interpretation. Results from different analysis techniques and literature reports need to be organized, processed, and interpreted to enable optimal decision-making for extracts prioritization. Here, we introduce Inventa, a computational tool that highlights the chemical novelty potential within extracts, considering untargeted mass spectrometry data, spectral annotation, and literature reports. Based on this information, Inventa calculates multiple scores that inform their chemical potential. Thus, Inventa has the potential to accelerate new natural products discovery. Inventa was applied to a set of plants from the Celastraceae family as a proof of concept. The Pristimera indica (Willd.) A.C.Sm roots extract was highlighted as a promising source of potentially novel compounds. Its phytochemical investigation resulted in the isolation and de novo characterization of thirteen new dihydro-{beta}-agarofuran sesquiterpenes, five of them presenting a new 9-oxodihydro-{beta}-agarofuran base scaffold.

Fungal infections pose a great threat to public health and there are limited antifungal medicaments. Streptomyces is an important source of antibiotics, represented by the clinical drug amphotericin B. The rapamycin-producer Streptomyces iranensis harbors an unparalleled Type I polyketide synthase, which codes for a novel antifungal macrolide alligamycin A (1), the structure of which was confirmed by NMR, MS, and X-ray crystallography. Alligamycin A harbors an undescribed carbon skeleton with 13 chiral centers, featuring a ({beta}-lactone moiety, a [6,6]-spiroketal ring, and an unprecedented 7-oxo-octylmalonyl-CoA extender unit incorporated by a potential novel crotonyl-CoA carboxylase/reductase. The ali biosynthetic gene cluster was confirmed through CRISPR-based gene editing. Alligamycin A displayed profound antifungal effects against numerous clinically relevant filamentous fungi, including Talaromyces and Aspergillus species. ({beta}-Lactone ring is essential for the antifungal activity and alligamycin B (2) with disruption in the ring abolished the antifungal effect. Proteomics analysis revealed alligamycin A potentially disrupted the integrity of fungal cell walls and induced the expression of stress-response proteins in Aspergillus niger. Alligamycins represent a new class of potential drug candidate to combat fungal infections.

Molecular recognition is a central component that confers detection specificity to all biosensors. The design and use of such molecules require consideration of properties including their affinity and selectivity, plus their ease of production and engineering, for downstream commercial purposes. Progesterone (P4), is a biomarker that is extensively for various diagnostic purposes. Examples include detection of P4 as an indicator of oestrus in cattle breeding, and ovulation in human IVF programs. P4 is also thought to promote strains of breast cancer, resulting in it being an environmental pollutant of interest. The present study focusses on in-silico molecular docking trials of P4 molecules with proteins such as antibodies and receptors. We describe the geometry of novel P4-binding pockets and predict key residues that favour high affinity and selectivity for P4. The in-silico molecular docking trials were performed on various mutants of an anti-P4 antibody that had lost their P4 specificity but retained selective recognition of steroids with structures closely related to cholesterol. Reverse-docking trials permitted the identification of novel scaffolds with favourable P4 binding properties. Future reports will validate the predictions of these studies through wet lab experiments. A further opportunity for this approach is to incorporate a scaffold functionality to permit binding of the protein or receptor to other molecules or sites within a biosensor electrode. These findings, and future studies, will assist in development of enhanced biosensing platforms with custom-designed P4 binders, aiding commercialisation using in-house developed reagents to meet IP requirements and minimise scaling costs. The steroid biotechnology market, valued at over $10 billion, also benefits from novel steroid binder designs, facilitating real-time steroid biomonitoring platforms for optimising steroid bioprocesses.

In the continuous pursuit of advanced cancer therapeutics, our research unveils the potential to selectively target Voltage-Dependent Anion-selective Channel isoform 1 (VDAC1), a pivotal component in cellular metabolism and apoptosis. VDAC1s role in metabolic rewiring and its subsequent prominence in many cancer types offer a unique intervention point. The incorporation of a systematic, in silico to in vitro methodology identified novel VA (VDAC-Antagonist) molecules with the capability to selectively bind to VDAC1, displaying a substantial specificity towards cancer cells while sparing healthy ones. This research first led to the revelation of a specialized VDAC1 pocket, which accommodates the binding of these VA molecules, thereby instigating a selective displacement of NADH. The coenzyme is a critical metabolic substrate, and its displacement ensues in notable mitochondrial distress and a reduction in cell proliferation, specifically in cancer cells. Furthermore, meticulous analysis using organoids derived from intrahepatic cholangiocarcinoma patients (iCCA) demonstrated a dose-dependent reduction in cell viability upon treatment with VA molecules, correlating with the findings from commercial cell lines. Interestingly, VA molecules significantly reduced cell viability and demonstrated a lower impact on healthy cells than conventional treatments like gemcitabine. This differential impact is possibly due to the elevated expression of VDAC1 in various cancer cell lines, rendering them more susceptible to metabolic disruptions induced by VA molecules. This endeavor uncovers a multifaceted approach to cancer treatment, involving meticulous targeting of metabolic gatekeepers like VDAC1 using novel entities, thereby paving the way for developing more selective and refined cancer therapeutics. The identified VA molecules, albeit in the nascent stages, represent promising candidates for further optimization and development, potentially revolutionizing treatment modalities in cancer therapy through precise metabolic interventions.

1.The gene SLC6A15 encodes the protein B0AT2, a transporter for neutral amino acids. It is highly expressed in the brain and has been associated with depression but little is otherwise know about its function. In this study, we identified the first inhibitors of this protein to pharmacologically investigate its function. The transporter activity was evaluated using a cellular uptake of the substrate 3H-proline. A miniaturized assay was developed and used for a High Throughput Screening (HTS) of 200,000 compounds. Hits were tested for cell toxicity and selectivity versus related transporters of the SLC6 family. The most promising inhibitors were validated by proline uptake and neurite outgrowth in primary hippocampal neurons. Of the 10 chemical scaffolds identified, a 1,5-benzodiazepine series had the most promising selectivity and structure-activity relationship (SAR) profile. The best compounds showed drug-like properties and inhibited B0AT2 with an IC50 of 250 nM both in SLC6A15-overexpressing HEK293 cells and primary neurons, with no detectable inhibition (> 80{micro}M) of SERT, DAT, GAT1, or NTT4/SLC6A17. These compounds also dose-dependently stimulated neurite outgrowth in primary neurons. The identified compounds are the first inhibitors of the amino acids transporter B0AT2/SLC6A15. Their potency, selectivity and physicochemical properties allow to target the transporter in relevant biological systems and to initiate new studies to understand its role and implication in diseases.

Homologation of amino acids, the addition or deletion of a methylene group onto their side chains, has the potential to increase the biostability and bioavailability of peptide natural products. The first enzyme in the homologation pathway, HphA, has been previously characterized and is substrate selective. Bioinformatics studies were used to identify amino acids in the active site of HphA, which may play a role in substrate selection, by comparison to homologous enzymes, homocitrate synthase (HCS) and 2-isopropylmalate synthase (IPMS). Single point mutants to five amino acid residues in the HphAs active site were created to mimic those of HCS and IPMS. Their activities were measured via time-course assays with the natural substrates for HCS and IPMS. Residue A73 was identified as important in the substrate specificity of HphA; therefore, six different additional mutations were generated and tested with nine substrates with various side chains. The HphA A73L mutant exhibited the highest activity compared to the other mutants, showing activity with counterparts of L-Tyr (HphA natural substrate), L-Val (IPMS natural substrate), L-Leu, L-Ser, L-Trp, and L-Asp. Kinetic assays were taken with HphA A73L with the active substrates and compared with kinetic data from HphA WT, HCS, and IPMS. These results demonstrated that the A73L mutation significantly relaxed the substrate specificity of HphA, indicating its amenability to engineering. This research will serve as the foundation for future metabolic engineering studies on the enzymatic homologation pathway of amino acids.

Molecular docking is a computational method used to predict the preferred binding orientation of one molecule to another when bound to each other to form an energetically stable complex. This approach has been widely used for early-stage small-molecule drug design as well as identifying suitable protein-based macromolecule residues for mutagenesis. Estimating binding free energy, based on docking interactions of protein to its ligand based on an appropriate scoring function is often critical for protein mutagenesis studies to improve the activity or alter the specificity of targeted enzymes. However, calculating docking free energy for a large number of protein mutants is computationally challenging and time-consuming. Here, we showcase an end-to-end computational workflow for predicting the binding energy of pNP-Xylose substrate docked within the substrate binding site for a large library of combinatorial mutants of an alpha-L-fucosidase (TmAfc, PDB ID-2ZWY) belonging to Thermotoga maritima glycosyl hydrolase (GH) family 29. Briefly, in silico combinatorial mutagenesis was performed for the top conserved residues in TmAfc as determined by running multiple sequence alignment against all GH29 family enzyme sequences downloaded from an in-house developed Carbohydrate-Active enZyme (CAZy) database retriever program. The binding energy was calculated through Autodock Vina with pNP-Xylose ligand docking with energy minimized TmAfc mutants, and the data was then used to train a neural network model which was also validated for model predictions using data from Autodock Vina. The current workflow can be adopted for any family of CAZymes to rapidly identify the effect of different mutations within the active site on substrate binding free energy to identify suitable targets for mutagenesis. We anticipate that this workflow could also serve as the starting point for performing more sophisticated and computationally intensive binding free energy calculations to identify targets for mutagenesis and hence optimize use of wet lab resources.

Candida antarctica lipase B (CALB) is widely used in biocatalysis with applications in plastics degradation and chemical synthesis. CALB is activated by hydrophobic matrices and, enigmatically, shows striking activation in polar, choline-based, Deep Eutectic Solvents (DES). Herein, we show that CALB activation and stabilisation by TAAs is caused by binding to cholines tetraalkylammonium (TAA) moiety. Several related TAA salts also caused CALB activation which was proportional to the hydrophobicity of their alkyl substituents. Remarkably, tetraoctylammonium bromide showed activation of [~]500% even at low micromolar levels. These TAA salts represent a new class of enzyme activator. Molecular modelling identified the alkylammonium binding location as a hydrophobic patch centred around Asp-145 of CALB. Binding at this site explains lipase activation in polar DES solvents and its relationship to other pathways of CALB activation. Herein, we also demonstrate that CALB, like many lipases, is activated by calcium. Intriguingly, mixed soluble activator experiments showed that calcium and choline bind to different CALB sites, suggesting a two-site model for CALB activation. These observations, along with previous findings, show that TAA activation is a widespread property of enzymes and constitutes a novel and potent means to enhance enzyme turnover and stability. HighlightsO_LICALB is activated by choline C_LIO_LISeveral tetraalkylammonium salts cause activation of CALB C_LIO_LIHyperactivation of CALB (5-fold) by tetraoctylammonium ions occurs at low micromolar concentrations. C_LIO_LITwo independent sites for CALB activation, by calcium and TAA ions, are identified C_LIO_LIActivation at the choline binding site stabilises CALB while calcium binding destabilises the enzyme C_LIO_LIA soluble activator is demonstrated, that can be used to probe the activation mechanism of CALB or other enzymes. C_LI

High prevalence of parasitic or bacterial infectious diseases in some world areas is due to multiple reasons, including a lack of an appropriate health policy, challenging logistics and poverty. The support to research and development of new medicines to fight infectious diseases is one of the sustainable development goals promoted by World Health Organization (WHO). In this sense, the traditional medicinal knowledge substantiated by ethnopharmacology is a valuable starting point for drug discovery. This work aims at the scientific validation of the traditional use of Piper species ("Cordoncillos") as firsthand anti-infectious medicines. For this purpose, we adapted a computational statistical model to correlate the LCMS chemical profiles of 54 extracts from 19 Piper species to their corresponding anti-infectious assay results based on 37 microbial or parasites strains. We mainly identified two groups of bioactive compounds (called features as they are considered at the analytical level and are not formally isolated). Group 1 is composed of 11 features being highly correlated to an inhibiting activity on 21 bacteria (principally Gram-positive strains), one fungus (C. albicans), and one parasite (Trypanosoma brucei gambiense). The group 2 is composed of 9 features having a clear selectivity on Leishmania (all strains, both axenic and intramacrophagic). Bioactive features in group 1 were identified principally in the extracts of Piper strigosum and P. xanthostachyum. In group 2, bioactive features were distributed in the extracts of 14 Piper species. This multiplexed approach provided a broad picture of the metabolome as well as a map of compounds putatively associated to bioactivity. To our knowledge, the implementation of this type of metabolomics tools aimed at identifying bioactive compounds has not been used so far.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiological agent responsible for the worldwide coronavirus disease 2019 (COVID-19) outbreak. Investigation has confirmed that polysaccharide heparan sulfate can bind to the spike protein and block SARS-CoV-2 infection. Theoretically, similar structure of nature polysaccharides may also have the impact on the virus. Indeed, some marine polysaccharide has been reported to inhibit SARS-Cov-2 infection in vitro, however the convinced targets and mechanism are still vague. By high throughput screening to target 3CLpro enzyme, a key enzyme that plays a pivotal role in the viral replication and transcription using nature polysaccharides library, we discover the mixture polysaccharide 375 from seaweed Ecklonia kurome Okam completely block 3Clpro enzymatic activity (IC50, 0.48 {micro}M). Further, the homogeneous polysaccharide 37502 from the 375 may bind to 3CLpro molecule well (kD value : 4.23 x 10-6). Very interestingly, 37502 also can potently disturb spike protein binding to ACE2 receptor (EC50, 2.01 {micro}M). Importantly, polysaccharide 375 shows good anti-SARS-CoV-2 infection activity in cell culture with EC50 values of 27 nM (99.9% inhibiting rate at the concentration of 20 {micro}g/mL), low toxicity (LD50: 136 mg/Kg on mice). By DEAE ion-exchange chromatography, 37501, 37502 and 37503 polysaccharides are purified from native 375. Bioactivity test show that 37501 and 37503 may impede SARS-Cov-2 infection and virus replication, however their individual impact on the virus is significantly less that of 375. Surprisingly, polysaccharide 37502 has no inhibition effect on SARS-Cov-2. The structure study based on monosaccharide composition, methylation, NMR spectrum analysis suggest that 375 contains guluronic acid, mannuronic acid, mannose, rhamnose, glucouronic acid, galacturonic acid, glucose, galactose, xylose and fucose with ratio of 1.86 : 9.56 : 6.81 : 1.69 : 1.00 : 1.75 : 1.19 : 11.06 : 4.31 : 23.06. However, polysaccharide 37502 is an aginate which composed of mannuronic acid (89.3 %) and guluronic acid (10.7 %), with the molecular weight (Mw) of 27.9 kDa. These results imply that mixture polysaccharides 375 works better than the individual polysaccharide on SARS-Cov-2 may be the cocktail-like polysaccharide synergistic function through targeting multiple key molecules implicated in the virus infection and replication. The results also suggest that 375 may be a potential drug candidate against SARS-CoV-2.

Commodity chemicals are high-demand chemicals, used by chemical industries to synthesise cocountless chemical products of daily use. For many of these chemicals, the main production process uses petroleum-based feedstocks. Concerns over these limited resources and their associated environmental problems, as well as mounting global pressure to reduce CO2 emissions have motivated efforts to find biochemical pathways capable of producing these chemicals. Advances in metabolic engineering have led to the development of technologies capable of designing novel biochemical pathways to commodity chemicals. Computational software tools, ReactPRED and RetroPath2.0 were utilised to design 49 novel pathways to produce benzene, phenol, and 1,2-propanediol -- all industrially important chemicals with limited biochemical knowledge. A pragmatic methodology for pathway curation was developed to analyse thousands and millions of pathways that were generated using the software. This method utilises publicly accessible biological databases, including MetaNetX, PubChem, and MetaCyc to analyse the generated outputs and assign EC numbers to the predicted reactions. The workflow described here for pathway generation and curation can be used to develop novel biochemical pathways to commodity chemicals from numerous starting compounds.

Medicinal plants serve as valuable resources for the isolation of endophytic fungi. Vinca minor is a well-known producer of important vinca alkaloids and emerges as a promising source of endophytic fungi with antibacterial potential and biosynthetic capacity. In this study, we isolated an endophytic fungus from V. minor and identified it as Cosmosporella sp. VM-42. To date, relatively little is known about this fungal genus. The ethyl acetate extract of this isolate selectively inhibited Gram-positive bacteria, such as methicillin-sensitive and methicillin-resistant Staphylococcus aureus (MSSA and MRSA). Therefore, we isolated the most abundant compound from the crude extract and identified it as nectriapyrone with MIC and MBC values ranging from 125 to 62.5 {micro}g/mL against MSSA and MRSA strains. We further sequenced and annotated the 39.07 Mb genome of the isolate, revealing that it encodes 9,842 protein-coding genes, including 415 genes for carbohydrate-active enzymes and various biosynthetic gene clusters. Our untargeted metabolomic analysis shows that the fungus produces various secondary metabolites, including cyclodepsipeptides, dimeric naphtho-{gamma}-pyrones, and macrolactones, which are known to have antifungal and antibacterial activities. In addition, we used small-molecule epigenetic modulators to activate the expression of silent biosynthetic gene clusters to broaden the chemical profile of Cosmosporella sp. VM-42. Taken together, we provide a first systematic analysis of Cosmosporella sp. VM-42, and our results show that it is a promising source of compounds with pharmacological potential against drug resistant bacteria. HighlightsO_LIFirst comprehensive study of Cosmosporella sp. VM-42 by genomics and metabolomics C_LIO_LIThe fungus produces chemically-diverse secondary metabolites with antibacterial activity C_LIO_LINectriapyrone, the main compound, shows bactericidal activity against MSSA and MRSA C_LIO_LISmall-molecule epigenetic modulators trigger production of putatively new secondary metabolites. C_LIO_LIOther secondary metabolites of Cosmosporella may present novel bioactivities. C_LI

The genus Cassia is a significant source of secondary metabolites that are physiologically active and come from several chemical classes. The current research deals with the isolation, spectroscopic elucidation (1D and 2D NMR spectroscopy) and enzymatic activity of fifteen known compounds as well as a new unidentified avenasterol derivative namely 21-methylene-24-ethylidene lophenol. The urease and {beta}-glucosidase inhibitory effects of these compounds were studied for the first time, and molecular docking studies were also performed to verify the structure-activity relationships. All the compounds evaluated towards urease showed higher inhibitory activity (1.224{+/-}0.43 < IC50 > 6.678{+/-}0.11 M) compared to standard thiourea (IC50 = 18.61{+/-}0.11 M). Molecular docking results revealed that compound 7 strongly inhibits urease due to the formation of a stable ligand-urease complex via hydrogen bonding, van der Waal and hydrophobic interactions. Formation of a favourable complex of 7 with the target enzyme gave a more negative docking score (-6.95 kcal/mol) than that of thiourea (-3.13 kcal/mol). Regarding the {beta}-glucosidase enzyme, all the compounds evaluated did not show activity except compound 1 which inhibited the latter with a percentage of inhibition of 82.6. These findings imply that this plant may be a contender for developing novel treatments for infectious disorders brought on by urease-producing bacteria.

O_SCPLOWLC_SCPLOW-DOPA extradiol dioxygenases (DODAs) catalyze the production of betalains and hygroaurins pigments. The sequence of the DODAs found in Caryophyllales and Basidiomycetes are not conserved, although betalains are produced both by plants and fungi. Here we revise the coding region of the dodA gene of fly agaric [Amanita muscaria (L.) Lam.] and describe an alternative start codon downstream that enables the heterologous expression of AmDODA, a promiscuous O_SCPLOWLC_SCPLOW-DOPA dioxygenase. AmDODA is 43-amino acid residues shorter than the recombinant DODA previously reported but catalyzes the formation of two isomeric seco-DOPAs that are the biosynthetic precursors of betalains and hygroaurins. The putative active site of AmDODA contains two distinct His-His-Glu motifs that can explain the dual cleavage of O_SCPLOWLC_SCPLOW-DOPA according to the mechanism proposed for non-heme iron-dependent dioxygenases. Upon addition of excess O_SCPLOWLC_SCPLOW-DOPA, both the betaxanthin and hygroaurin adducts of O_SCPLOWLC_SCPLOW-DOPA are produced. The kinetic parameters of enzymatic catalysis at pH 8.5 are similar to those reported for other O_SCPLOWLC_SCPLOW-DOPA dioxygenases. The rate constants for the conversion of O_SCPLOWLC_SCPLOW-DOPA into the betalamic acid and muscaflavin were estimated by kinetic modelling allowing the proposal of a mechanism of pigment formation. These results contribute to understanding the biosynthesis of bacterial, fungal and plant pigments, for the biotechnological production of hygroaurins, and for the development of more promiscuous dioxygenases for environmental remediation.

Caenorhabditis elegans is an ideal model organism to study the xenobiotic detoxification pathways of various natural and synthetic toxins. One toxin shown to cause death in C. elegans is 1-hydroxyphenazine (1-HP), a molecule produced by the bacterium Pseudomonas aeruginosa. We previously showed that the median lethal dose (LD50) for 1-HP in C elegans is 179 M in PD1074 and between 150-200 M in N2 (C. elegans lab strain). We also showed that C. elegans detoxifies 1-HP by glycosylation by adding one, two, or three glucose molecules in N2 worms. This study tested whether UDP-glycosyltransferase (ugt) genes play a role in 1-HP detoxification. We show that ugt-23 and ugt-49 knockout mutants are more sensitive to 1-HP. Our data also show that ugt-23 knockout mutants produce reduced amounts of the trisaccharide sugars, while the ugt-49 knockout mutants produce reduced amounts of all 1-HP derivatives except for the glucopyranosyl product. We have also characterized the structure of the trisaccharide sugar phenazine structures made by C. elegans and show that one of the sugar modifications contains an N-acetylglucosamine (GlcNAc) in place of glucose. This implies broad specificity regarding UGT function and the role of genes other than ogt-1 in adding GlcNAc, at least in small-molecule detoxification.