Synthesis of Cyclopeptides Analogues of Natural Products and Evaluation as Herbicides and Inhibitors of Cyanobacteria.

Natural products derived from plants or microorganisms have been considered as eco-friendly herbicides with application in crop protection. Several natural cyclopeptides have been reported as herbicides, while others have been identified as inhibitors of cyanobacteria. In this work, the syntheses of cyclotetrapeptides and cyclopentapeptides analogues of natural products were successfully performed by solid-phase peptide synthesis of their linear precursor and solution-phase macrolactamization. Four of the obtained peptides and cyclopeptides present phytotoxicity with more than 70% of radicle growth inhibition at 67 µg/mL. In addition, evaluation of 20 peptides and cyclopeptides, as inhibitors of cyanobacteria, rendered five active compounds that reduced the concentration of microcystins in the culture medium.

Reverse Phase HPLC Methodology for the Determination of Bay K8644.

Following ICH guidelines for analytical validation, we report a common C18 column stability indicating isocratic reverse phase high performance liquid chromatography method for the determination of the ion channel modulator Bay K8644. Two main forced degradation products and a minor impurity were also tentatively identified by Mass Spectrometry. The mobile phase consisted of a 50/50 acetonitrile/buffer mixture at a flow rate of 2 mL/min. Mean retention time for Bay K8644 was 3.030 minutes. Excellent linearity (r = 0.9998) was achieved in the range 0.10-1.40 µg/mL at 274 nm wavelength. Analytical limits were 16.56 ± 1.04 ng/mL for detection and 55.21 ± 3.48 ng/mL for quantitation respectively. Accuracy and precision studies showed good results (95-105%). Robustness was assessed by varying ±3%, both temperature and flow rate. Five different stress conditions were applied to assess Bay K8644's stability. Only basic and photolytic treatments yielded degradation products, both correctly resolved in a total runtime of 4 minutes. In conclusion, we developed a fast, simple, sensitive, accurate, precise, reliable and stability indicating method for detecting/quantifying Bay K8644, and tentatively characterized its main impurities/forced degradation products.

An antibiotic produced by Pseudomonas fluorescens CFBP2392 with antifungal activity against Rhizoctonia solani.

Pseudomonas fluorescens CFBP2392 has been recognized as a potential biocontrol agent due to its ability to suppress damping-off and root rot disease. This isolate has antibacterial activity in vitro as many other strains from the Pseudomonas fluorescens complex. In this work, the antibacterial and antifungal activity of the strain were explored. Dual culture assays evidenced the antifungal activity of the strain against different phytopathogens: Alternaria sp., Pythium ultimun, Fusarium oxysporum, and Rhizoctonia solani. Purification of an antifungal fraction was performed by preparative HPLC from the chemical extraction of growth media. The fraction showed altered R. solani growth and ultrastructure. Transmission electron microscopy revealed the purified compound induced hypertrophied mitochondria, membranous vesicles, and a higher number of vacuoles in R. salani cytoplasm. In addition, co-cultivation of P. fluorescens CFBP2392 with R. solani resulted in an enlarged and deformed cell wall. To gain genomic insights on this inhibition, the complete genome of P. fluorescens CFBP2392 was obtained with Oxford Nanopore technology. Different biosynthetic gene clusters (BGCs) involved in specialized metabolites production including a lokisin-like and a koreenceine-like cluster were identified. In accordance with the putative BGCs identified, sequence phylogeny analysis of the MacB transporter in the lokisin-like cluster further supports the similarity with other transporters from the amphisin family. Our results give insights into the cellular effects of the purified microbial metabolite in R. solani ultrastructure and provide a genomic background to further explore the specialized metabolite potential.

Synthesis of Cyclotetrapeptides Analogues to Natural Products as Herbicides.

The synthesis of cyclotetrapeptides analogues of the natural products tentoxin and versicotide D was achieved in good yield by solid phase peptide synthesis (SPPS) of their linear precursors and solution phase cyclization. All the cyclopeptides and several open precursors were evaluated as herbicides. Five cyclopeptides and five lineal peptides showed a significant inhibition (>70%) of Ryegrass seed's radicle growth at 67 µg/mL. The evaluation at lower concentrations (4−11 µM) indicates two cyclopeptides analogs of tentoxin, which present one (N-Methyl-d-Phe), and two N-MeAA (N-Methyl-Ala and N-Methyl-Phe), respectively, as the most active of them, showing remarkable phytotoxic activity. In two cases, the open precursors are as active as their corresponding cyclopeptide. However, many linear peptides are inactive and their cyclization derivatives showed herbicidal activity. In addition, two cyclopeptide analogues of versicotide D showed more improved activity than the natural product. The results indicate that the peptide sequence, the amino acid stereochemistry and the presence of N-methyl group have important influence on the phytotoxic activity. Moreover, several compounds could be considered as lead candidates in the development of bioherbicides.

New Insights into the Chemical Composition of Ayahuasca.

Ayahuasca is a psychedelic beverage originally from the Amazon rainforest used in different shamanic settings for medicinal, spiritual, and cultural purposes. It is prepared by boiling in water an admixture of the Amazonian vine Banisteriopsis caapi, which is a source of ß-carboline alkaloids, with plants containing N,N-dimethyltryptamine, usually Psychotria viridis. While previous studies have focused on the detection and quantification of the alkaloids present in the drink, less attention has been given to other nonalkaloid components or the composition of the solids suspended in the beverage, which may also affect its psychoactive properties. In this study, we used nuclear magnetic resonance (NMR) and liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) to study the composition of ayahuasca samples, to determine their alkaloid qualitative and quantitative profiles, as well as other major soluble and nonsoluble components. For the first time, fructose was detected as a major component of the samples, while harmine (a ß-carboline previously described as an abundant alkaloid in ayahuasca) was found to be present in the solids suspended in the beverage. In addition, N,N-dimethyltryptamine (DMT), harmine, tetrahydroharmine, harmaline, and harmol were identified as the major alkaloids present in extracts of all samples. Finally, a novel, easy, and fast method using quantitative NMR was developed and validated to simultaneously quantify the content of these alkaloids found in each ayahuasca sample.

Neuroprotective effects of violacein in a model of inherited amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by progressive death of motor neurons and muscle atrophy, with defective neuron-glia interplay and emergence of aberrant glial phenotypes having a role in disease pathology. Here, we have studied if the pigment violacein with several reported protective/antiproliferative properties may control highly neurotoxic astrocytes (AbAs) obtained from spinal cord cultures of symptomatic hSOD1G93A rats, and if it could be neuroprotective in this ALS experimental model. At concentrations lower than those reported as protective, violacein selectively killed aberrant astrocytes. Treatment of hSOD1G93A rats with doses equivalent to the concentrations that killed AbAs caused a marginally significant delay in survival, partially preserved the body weight and soleus muscle mass and improved the integrity of the neuromuscular junction. Reduced motor neuron death and glial reactivity was also found and likely related to decreased inflammation and matrix metalloproteinase-2 and -9. Thus, in spite that new experimental designs aimed at extending the lifespan of hSOD1G93A rats are needed, improvements observed upon violacein treatment suggest a significant therapeutic potential that deserves further studies.

Production and antiproliferative effect of violacein, a purple pigment produced by an Antarctic bacterial isolate.

We studied the production and the potential use of a purple-pigment produced by an Antarctic bacterial isolate. This pigment was identified as violacein, a metabolite produced by many bacterial strains and reported that it has antiproliferative activity in many cell lines. We analyzed the effect of temperature and the composition of the growth medium on pigment production, achieving the highest yield at 20 °C in Tryptic Soy Broth medium supplemented with 3.6 g/L glucose. We doubled the yield of the pigment production when the process was scaled up in a 5 L bioreactor (77 mg/L of crude pigment). The pigment was purified and identified by mass spectrometry (DI-EI-MS) and Nuclear Magnetic Resonance (NMR) spectroscopy as violacein. We performed survival assays that showed that the pure pigment has antiproliferative activity and sensitize HeLa cells (cervix cell carcinoma) to cisplatin. Besides, the pigment did not show genotoxic activity in HeLa cells as found performing micronucleus assays. These results suggest that this pigment may be used as anticancer or sensitizer to cisplatin drug in cervix cancer.

Identification of a Potential Zika Virus Inhibitor Targeting NS5 Methyltransferase Using Virtual Screening and Molecular Dynamics Simulations.

The NS5 methyltransferase (MTase) has been reported as an attractive molecular target for antivirals discovery against the Zika virus (ZIKV). Here, we report structure-based virtual screening of 42 390 structures from the Development Therapeutics Program (DTP) AIDS Antiviral Screen Database. Among the docked compounds, ZINC1652386 stood out due to its high affinity for MTase in comparison to the cocrystallized ligand MS2042, which interacts with the Asp146 residue in the MTase binding site by hydrogen bonding. Subsequent molecular dynamics simulations predicted that this compound forms a stable complex with MTase within 50 ns. Thus, ZINC1652386 may represent a promising ZIKV methyltransferase inhibitor.

First total synthesis of versicotide A, B and C.

The syntheses of versicotides A-C, natural products containing anthranilic acid and NMe-Ala, were achieved by solid phase peptide synthesis on 2-chlorotrityl resin followed by solution phase macrocyclization. Using an oxyma additive, the difficult coupling reactions to the deactivated aromatic amine of o-aminobenzoic acid, were performed in high yield, avoiding anthranilic rearrangements or side reactions.

Preparation and mechanistic studies of 2-substituted Bisthiazolidines by imine exchange.

Bisthiazolidines (BTZ) are bicyclic compounds considered penicillin analogs that inhibit the full range of Metallo-ß-Lactamases (MBLs) and potentiate ß-lactam activity against resistant bacteria. Herein we present a new methodology to prepare 2-substituted bisthiazolidines by aldehyde exchange. Thirteen new bisthiazolidines were prepared using this methodology, with yields ranged from 31 to 75%. The reaction is based on in situ imines formation, which are able to exchange side chains. The reaction intermediates were studied based on NMR experiments and a key imine 1b-II could be detected in the reaction mixture. Furthermore, a DFT computational analysis was performed to gain insights into the reaction mechanism, allowing us to unveil the different pathways and their activation barriers within the synthetic route. The results suggest that the most favorable route involve the formation of the thiazolidine 1b-III by i) a N-assisted N-C bond cleavage, and ii) a thiol-mediated 5 endo-trig cyclization followed by a C-N bond cleavage. In contrast with previously reported evidence, the imine metathesis was discarded as a plausible pathway. Finally, the reaction of 1b-III with aldehyde 2a leads to bicycle 4a via the iminium ion 1b-V.

Identification of Zika Virus NS2B-NS3 Protease Inhibitors by Structure-Based Virtual Screening and Drug Repurposing Approaches.

The NS2B-NS3 protease has been identified as an attractive target for drug development against Zika virus (ZIKV) and combined drug repurposing and structure-based virtual screening has improved the development of antiviral drugs. In this study, we performed a structure-based virtual screening of 1861 Food and Administration (FDA) approved drugs available in DrugBank by the selection and docking validation of crystal structure of ZIKV NS2B-NS3 protease (PDB ID 5H4I ) using Glide and DOCK 6 software. The antihistaminic chlorcyclizine (Grid score -24.8 kcal/mol) exhibited the most promising interaction with NS2B-NS3 protease in comparison to crystallography ligand (Grid score -15.6 kcal/mol) by interaction to Tyr161 by hydrophobic interactions in the binding site of NS2B-NS3 which is recognized as an important amino acid in substrate molecular recognition. Cytotoxicity and global antiviral activity assay in Vero cells by MTT method showed that chlorcyclizine reduced the ZIKV induced cytopathic effect (EC50 of 69.0 ± 7.3 µM and SI = 1.9), and explicit molecular dynamics simulations implemented on a NAMD program indicated great stability of chlorcyclizine in protease binding site, suggesting the repurposing of chlorcyclizine as a promising finding in anti-ZIKV drug development.

Validating biochemical features at the genome level in the Antarctic bacterium Hymenobacter sp. strain UV11.

We present experimental data that complement and validate some biochemical features at the genome level in the UVC-resistant Antarctic bacterium Hymenobacter sp. UV11 strain. The genome was sequenced, assembled and annotated. It has 6 096 246 bp, a GC content of 60.6% and 5155 predicted genes. The secretome analysis, by combining in silico predictions with shotgun proteomics data, showed that UV11 strain produces extracellular proteases and carbohydrases with potential biotechnological uses. We observed the formation of outer membrane vesicles, mesosomes and carbon-storage compounds by using transmission electron microscopy. The in silico analysis of the genome revealed the presence of genes involved in the metabolism of glycogen-like molecules and starch. By HPLC-UV-Vis analysis and 1H-NMR spectra, we verified that strain UV11 produces xanthophyll-like carotenoids such as 2'-hydroxyflexixanthin, and the in silico analysis showed that this bacterium has genes involved in the biosynthesis of cathaxanthin, zeaxanthin and astaxanthin. We also found genes involved in the repair of UV-damaged DNA such as a photolyase, the nucleotide excision repair system and the production of ATP-dependent proteases that are important cellular components involved in the endurance to physiological stresses. This information will help us to better understand the ecological role played by Hymenobacter strains in the extreme Antarctic environment.

Identification and structural characterization of serobactins, a suite of lipopeptide siderophores produced by the grass endophyte Herbaspirillum seropedicae.

Herbaspirillum seropedicae Z67 is a diazotrophic endophyte able to colonize the interior of many economically relevant crops such as rice, wheat, corn and sorghum. Structures of siderophores produced by bacterial endophytes have not yet been elucidated. The aim of this work was to identify and characterize the siderophores produced by this bacterium. In a screening for mutants unable to produce siderophores we found a mutant that had a transposon insertion in a non-ribosomal peptide synthase (NRPS) gene coding for a putative siderophore biosynthetic enzyme. The chemical structure of the siderophore was predicted using computational genomic tools. The predicted structure was confirmed by chemical analysis. We found that siderophores produced by H. seropedicae Z67 are a suite of amphiphilic lipopeptides, named serobactin A, B and C, which vary by the length of the fatty acid chain. We also demonstrated the biological activity of serobactins as nutritional iron sources for H. seropedicae. These are the first structurally described siderophores produced by endophytic bacteria.

Cytotoxic, virucidal, and antiviral activity of South American plant and algae extracts.

Herpes simplex virus type 1 (HSV-1) infection has a prevalence of 70% in the human population. Treatment is based on acyclovir, valacyclovir, and foscarnet, three drugs that share the same mechanism of action and of which resistant strains have been isolated from patients. In this aspect, innovative drug therapies are required. Natural products offer unlimited opportunities for the discovery of antiviral compounds. In this study, 28 extracts corresponding to 24 plant species and 4 alga species were assayed in vitro to detect antiviral activity against HSV-1. Six of the methanolic extracts inactivated viral particles by direct interaction and 14 presented antiviral activity when incubated with cells already infected. Most interesting antiviral activity values obtained are those of Limonium brasiliense, Psidium guajava, and Phyllanthus niruri, which inhibit HSV-1 replication in vitro with 50% effective concentration (EC(50)) values of 185, 118, and 60 µg/mL, respectively. For these extracts toxicity values were calculated and therefore selectivity indexes (SI) obtained. Further characterization of the bioactive components of antiviral plants will pave the way for the discovery of new compounds against HSV-1.

Thiazole and oxazole alkaloids: isolation and synthesis.

Thiazoles, oxazole and their corresponding reduced derivatives, thiazolines and oxazolines, are found in marine sources exhibiting significant biological activities. The isolation, synthetic, and biological studies of these natural products, covering literature from January 2007 to June 2010, are summarized.

Bisabolanes from the red alga Laurencia scoparia.

Three novel halogenated beta-bisabolene sesquiterpenoids (1-3), together with two know triquinane alcohol sesquiterpenes (6 and 7), were isolated from the red alga Laurencia scoparia and their structures elucidated by spectroscopic methods. Single-crystal X-ray crystallography allowed us to confirm the structure of 1 as well as to determine the absolute configuration of all stereocenters. To the best of our knowledge, the isolation of beta-bisabolenes from the genus Laurencia has no precedent in the literature. Compound 1 showed weak in vitro anthelmintic activity against parasitant stage (L4) Nippostrongilus brasiliensis.