Pineapple Lectin AcmJRL Binds SARS-CoV-2 Spike Protein in a Carbohydrate-Dependent Fashion.

The highly glycosylated spike protein of SARS-CoV-2 is essential for infection and constitutes a prime target for antiviral agents and vaccines. The pineapple-derived jacalin-related lectin AcmJRL is present in the medication bromelain in significant quantities and has previously been described to bind mannosides. Here, we performed a large ligand screening of AcmJRL by glycan array analysis, quantified the interaction with carbohydrates and validated high-mannose glycans as preferred ligands. Because the SARS-CoV-2 spike protein was previously reported to carry a high proportion of high-mannose N-glycans, we tested the binding of AcmJRL to the recombinantly produced extraviral domain of spike protein. We could demonstrate that AcmJRL binds the spike protein with a low-micromolar KD in a carbohydrate-dependent fashion.

Persicamidines-Unprecedented Sesquarterpenoids with Potent Antiviral Bioactivity against Coronaviruses.

A new family of highly unusual sesquarterpenoids (persicamidines A-E) exhibiting significant antiviral activity was isolated from a newly discovered actinobacterial strain, Kibdelosporangium persicum sp. nov., collected from a hot desert in Iran. Extensive NMR analysis unraveled a hexacyclic terpenoid molecule with a modified sugar moiety on one side and a highly unusual isourea moiety fused to the terpenoid structure. The structures of the five analogues differed only in the aminoalkyl side chain attached to the isourea moiety. Persicamidines A-E showed potent activity against hCoV-229E and SARS-CoV-2 viruses in the nanomolar range together with very good selectivity indices, making persicamidines promising as starting points for drug development.

Characterization of bromelain indicates a molar excess of inhibitor vs. enzyme molecules, a Jacalin-like lectin and Maillard reaction products.

The phytotherapeutic bromelain is a heterogeneous protein mixture, extracted from pineapple stem, with high proteolytic activity based on cysteine proteases. Its global protein chemical composition was analyzed qualitatively and quantitatively by SDS-PAGE and RP-HPLC. A SDS-PAGE method with elaborate sample pretreatment was developed, to cope with the bromelain's self-digestion properties and the hypothetical disulfide scrambling during electrophoresis. Both can produce misleading results, if not considered. RP-HPLC was applied for its high separation power for bromelain proteinaceous compounds. A peak identification and assignment to different protein classes in bromelain was done by enzyme kinetics and MS. The method was successfully applied for the quantitative determination of the molar ratio between inhibitor and enzyme and resulted to be approximately 3:2. Bromelain contains, from a molar point of view, inhibitor molecules as major component, which thus might be considered as a natural pharmaceutical excipient in Bromelain, because it protects the enzymes against autolysis. We described two methods to separate the inhibitor fraction from the enzyme fraction, RP-HPLC and size exclusion chromatography. A pineapple derived Jacalin-like-lectin, herein called 'Anlec', was identified and quantified by RP-HPLC-MS in bromelain and its content was determined to be 5%, related to all proteins in bromelain. Anlec binds specifically to mannose-containing glycans and is discussed in literature to possess anti-HIV medical potential. Bromelain could therefore be a possible and economic source for the production of Anlec. An isolation strategy of Anlec from bromelain, in high purity, is shown in this work. The presented RP-HPLC results are comprehensive in chemical information, and the method is expedient to provide appropriate bromelain protein isolations but also to accomplish quality control, covering all relevant protein components. It is furthermore shown, that proteins in bromelain may react with reducing sugars in a Maillard reaction to form glycated proteins. Maillard reaction products in bromelain are detected and characterized and could be responsible for the limited stability and storage times at room temperature of bromelain. Even the active center thiol group could be potentially glycated.

Activation of the NLRP3 Inflammasome by Hyaboron, a New Asymmetric Boron-Containing Macrodiolide from the Myxobacterium Hyalangium minutum.

A Natural Compound Library containing myxobacterial secondary metabolites was screened in murine macrophages for novel activators of IL-1ß maturation and secretion. The most potent of three hits in total was a so far undescribed metabolite, which was identified from the myxobacterium Hyalangium minutum strain Hym3. While the planar structure of 1 was elucidated by high resolution mass spectrometry and NMR data yielding an asymmetric boron containing a macrodiolide core structure, its relative stereochemistry of all 20 stereocenters of the 42-membered ring was assigned by rotating frame Overhause effect spectroscopy correlations, 1H,1H, and 1H,13C coupling constants, and by comparison of 13C chemical shifts to those of the structurally related metabolites tartrolon B-D. The absolute stereochemistry was subsequently assigned by Mosher's and Marfey's methods. Further functional studies revealed that hyaboron and other boronated natural compounds resulted in NLRP3 inflammasome dependent IL-1ß maturation, which is most likely due to their ability to act as potassium ionophores. Moreover, besides its inflammasome-stimulatory activity in human and mouse cells, hyaboron (1) showed additional diverse biological activities, including antibacterial and antiparasitic effects.

Improved riboflavin production with Ashbya gossypii from vegetable oil based on 13C metabolic network analysis with combined labeling analysis by GC/MS, LC/MS, 1D, and 2D NMR.

The fungus Ashbya gossypii is an important industrial producer of riboflavin, i.e. vitamin B2. In order to meet the constantly increasing demands for improved production processes, it appears essential to better understand the underlying metabolic pathways of the vitamin. Here, we used a highly sophisticated set-up of parallel 13C tracer studies with labeling analysis by GC/MS, LC/MS, 1D, and 2D NMR to resolve carbon fluxes in the overproducing strain A. gossypii B2 during growth and subsequent riboflavin production from vegetable oil as carbon source, yeast extract, and supplemented glycine. The studies provided a detailed picture of the underlying metabolism. Glycine was exclusively used as carbon-two donor of the vitamin's pyrimidine ring, which is part of its isoalloxazine ring structure, but did not contribute to the carbon-one metabolism due to the proven absence of a functional glycine cleavage system. The pools of serine and glycine were closely connected due to a highly reversible serine hydroxymethyltransferase. Transmembrane formate flux simulations revealed that the one-carbon metabolism displayed a severe bottleneck during initial riboflavin production, which was overcome in later phases of the cultivation by intrinsic formate accumulation. The transiently limiting carbon-one pool was successfully replenished by time-resolved feeding of small amounts of formate and serine, respectively. This increased the intracellular availability of glycine, serine, and formate and resulted in a final riboflavin titer increase of 45%.

Correlating chemical diversity with taxonomic distance for discovery of natural products in myxobacteria.

Some bacterial clades are important sources of novel bioactive natural products. Estimating the magnitude of chemical diversity available from such a resource is complicated by issues including cultivability, isolation bias and limited analytical data sets. Here we perform a systematic metabolite survey of ~2300 bacterial strains of the order Myxococcales, a well-established source of natural products, using mass spectrometry. Our analysis encompasses both known and previously unidentified metabolites detected under laboratory cultivation conditions, thereby enabling large-scale comparison of production profiles in relation to myxobacterial taxonomy. We find a correlation between taxonomic distance and the production of distinct secondary metabolite families, further supporting the idea that the chances of discovering novel metabolites are greater by examining strains from new genera rather than additional representatives within the same genus. In addition, we report the discovery and structure elucidation of rowithocin, a myxobacterial secondary metabolite featuring an uncommon phosphorylated polyketide scaffold.

Heterologous expression of the plant cysteine protease bromelain and its inhibitor in Pichia pastoris.

Expression of proteases in heterologous hosts remains an ambitious challenge due to severe problems associated with digestion of host proteins. On the other hand, proteases are broadly used in industrial applications and resemble promising drug candidates. Bromelain is an herbal drug that is medicinally used for treatment of oedematous swellings and inflammatory conditions and consists in large part of proteolytic enzymes. Even though various experiments underline the requirement of active cysteine proteases for biological activity, so far no investigation succeeded to clearly clarify the pharmacological mode of action of bromelain. The potential role of proteases themselves and other molecules of this multi-component extract currently remain largely unknown or ill defined. Here, we set out to express several bromelain cysteine proteases as well as a bromelain inhibitor molecule in order to gain defined molecular entities for subsequent studies. After cloning the genes from its natural source Ananas comosus (pineapple plant) into Pichia pastoris and subsequent fermentation and purification, we obtained active protease and inhibitor molecules which were subsequently biochemically characterized. Employing purified bromelain fractions paves the way for further elucidation of pharmacological activities of this natural product. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:54-65, 2017.

Discovery of the first small-molecule CsrA-RNA interaction inhibitors using biophysical screening technologies.

AIM: CsrA is a global post-transcriptional regulator protein affecting mRNA translation and/or stability. Widespread among bacteria, it is essential for their full virulence and thus represents a promising anti-infective drug target. Therefore, we aimed at the discovery of CsrA-RNA interaction inhibitors. Results & methodology: We followed two strategies: a screening of small molecules (A) and an RNA ligand-based approach (B). Using surface plasmon resonance-based binding and fluorescence polarization-based competition assays, (A) yielded seven small-molecule inhibitors, among them MM14 (IC50 of 4 µM). (B) resulted in RNA-based inhibitor GGARNA (IC50 of 113 µM). CONCLUSION: The first small-molecule inhibitors of the CsrA-RNA interaction were discovered exhibiting micromolar affinities. These hits represent tools to investigate the effects of CsrA-RNA interaction inhibition on bacterial virulence.

Effects of Halide Ions on the Carbamidocyclophane Biosynthesis in Nostoc sp. CAVN2.

In this study, the influence of halide ions on [7.7]paracyclophane biosynthesis in the cyanobacterium Nostoc sp. CAVN2 was investigated. In contrast to KI and KF, supplementation of the culture medium with KCl or KBr resulted not only in an increase of growth but also in an up-regulation of carbamidocyclophane production. LC-MS analysis indicated the presence of chlorinated, brominated, but also non-halogenated derivatives. In addition to 22 known cylindrocyclophanes and carbamidocyclophanes, 27 putative congeners have been detected. Nine compounds, carbamidocyclophanes M-U, were isolated, and their structural elucidation by 1D and 2D NMR experiments in combination with HRMS and ECD analysis revealed that they are brominated analogues of chlorinated carbamidocyclophanes. Quantification of the carbamidocyclophanes showed that chloride is the preferably utilized halide, but incorporation is reduced in the presence of bromide. Evaluation of the antibacterial activity of 30 [7.7]paracyclophanes and related derivatives against selected pathogenic Gram-positive and Gram-negative bacteria exhibited remarkable effects especially against methicillin- and vancomycin-resistant staphylococci and Mycobacterium tuberculosis. For deeper insights into the mechanisms of biosynthesis, the carbamidocyclophane biosynthetic gene cluster in Nostoc sp. CAVN2 was studied. The gene putatively coding for the carbamoyltransferase has been identified. Based on bioinformatic analyses, a possible biosynthetic assembly is discussed.

Antibiotics. Targeting DnaN for tuberculosis therapy using novel griselimycins.

The discovery of Streptomyces-produced streptomycin founded the age of tuberculosis therapy. Despite the subsequent development of a curative regimen for this disease, tuberculosis remains a worldwide problem, and the emergence of multidrug-resistant Mycobacterium tuberculosis has prioritized the need for new drugs. Here we show that new optimized derivatives from Streptomyces-derived griselimycin are highly active against M. tuberculosis, both in vitro and in vivo, by inhibiting the DNA polymerase sliding clamp DnaN. We discovered that resistance to griselimycins, occurring at very low frequency, is associated with amplification of a chromosomal segment containing dnaN, as well as the ori site. Our results demonstrate that griselimycins have high translational potential for tuberculosis treatment, validate DnaN as an antimicrobial target, and capture the process of antibiotic pressure-induced gene amplification.

Elevated free cholesterol in a p62 overexpression model of non-alcoholic steatohepatitis.

AIM: To characterize how insulin-like growth factor 2 (IGF2) mRNA binding protein p62/IMP2-2 promotes steatohepatitis in the absence of dietary cholesterol. METHODS: Non-alcoholic steatohepatitis (NASH) was induced in wild-type mice and in mice overexpressing p62 specifically in the liver by feeding the mice a methionine and choline deficient (MCD) diet for either two or four weeks. As a control, animals were fed a methionine and choline supplemented diet. Serum triglycerides, cholesterol, glucose, aspartate aminotransferase and alanine transaminase were determined by standard analytical techniques. Hepatic gene expression was determined by real-time reverse transcription-polymerase chain reaction. Generation of reactive oxygen species in liver tissue was quantified as thiobarbituric acid reactive substances using a photometric assay and malondialdehyde as a standard. Tissue fatty acid profiles and cholesterol levels were analyzed by gas chromatography-mass spectrometry after hydrolysis. Hepatocellular iron accumulation was determined by Prussian blue staining in paraffin-embedded formalin-fixed tissue. Filipin staining on frozen liver tissue was used to quantify hepatic free cholesterol levels. Additionally, nuclear localization of the nuclear factor kappa B (NF-κB) subunit p65 was examined in frozen tissues. RESULTS: Liver-specific overexpression of the insulin-like growth factor 2 mRNA binding protein 2-2 (IGF2BP2-2/IMP2-2/p62) induces steatosis with regular chow and amplifies NASH-induced fibrosis in the MCD mouse model. Activation of NF-κB and expression of NF-κB target genes suggested an increased inflammatory response in p62 transgenic animals. Analysis of hepatic lipid composition revealed an elevation of monounsaturated fatty acids as well as increased hepatic cholesterol. Moreover, serum cholesterol was significantly elevated in p62 transgenic mice. Dietary cholesterol represents a critical factor for the development of NASH from hepatic steatosis. Filipin staining revealed increased free cholesterol in p62 transgenic livers, which were not diet-derived. The mRNA levels of the rate-limiting enzyme for cholesterol synthesis 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMG-CoA reductase or HMGCR) were not significantly upregulated, potentially due to increased cholesterol biosynthesis via elevated sterol regulatory element binding transcription factor 2 (SREBF2) gene expression and increased iron deposition in transgenic animals. CONCLUSION: This study provides evidence that p62/IGF2BP2-2 drives the progression of NASH through elevation of hepatic iron deposition and increased production of hepatic free cholesterol.

Discovery and biological activity of new chondramides from Chondromyces sp.

Myxobacteria have proven to be highly valuable sources of natural products, as they produce a variety of secondary metabolites with unique structures and often new modes of action. In this study, high-content screening is demonstrated to be a convenient tool for bioactivity-guided isolation of natural products from crude bacterial extracts. By the application of focused, image-based screens we were able to identify over 30 novel chondramide derivatives from Chondromyces sp. MSr9030, some of which were present in only minute amounts. These cyclic depsipeptides were shown to target actin filaments with a similar binding mode to that of the mushroom toxin phalloidin. Fermentations of the myxobacterial strain were carried out under improved cultivation conditions, and supplementation of the culture broth with potassium bromide afforded the production of brominated analogues that are superior (in terms of biological activity) to all chondramides described to date. Initial biological profiling of 11 new derivatives in comparison to the reference compounds (chondramides A-C) showed that bromo-chondramide C3 and propionyl-bromo-chondramide C3 are the most active in cell-based studies, with GI50 values on human cancer cell lines in the low nanomolar range. Given that these brominated C3 analogues were also less potent on noncancerous human cells (by a factor of 2 to 4 in comparison to cancer cell lines), our results can aid further structure-activity relationship-guided development of chondramides, either as molecular probes or pharmaceutical agents.

Activity-guided screening of bioactive natural compounds implementing a new glucocorticoid-receptor-translocation assay and detection of new anti-inflammatory steroids from bacteria.

Using an in vitro cell-based assay in a flow-design, we have applied activity-guided screening to search for new bioactive compounds isolated from microorganisms. A first assay employs the stable expression of nuclear factor kappa B (NF-κB) while a second assay utilizes the glucocorticoid receptor (GR) coupled to green fluorescent protein. A specialized assay was implemented for both the translocation of NF-κB and to inhibit the translocation of cytokine-mediated NF-κB. In addition, we developed in a wide palette of cell lines used for a highly specialized GR-translocation assay to detect anti-inflammatory effects. This approach demonstrates the straight-forward combination of cell-based assays arranged with an automated fluorescence microscope. This allows for the direct sorting of extracts which are acting in a pharmaceutically interesting way. Initial results using this technique have led to the detection of new anti-inflammatory steroids from bacterial crude extracts.

Intracranial ependymoma: long-term results in a series of 21 patients treated with stereotactic (125)iodine brachytherapy.

BACKGROUND: We evaluated the long-term outcome in patients harboring intracranial ependymomas treated with interstitial brachytherapy (IBT). METHODS: Twenty-one patients (M/F = 9/12; median age: 29 years; range: 8-70 years), diagnosed with intracranial ependymoma (1 WHO I, 11 WHO II, 9 WHO III) were treated with IBT using stereotactically implanted (125)Iodine seeds between 1987 and 2010, either primarily, as adjuvant therapy following incomplete resection, or as salvage treatment upon tumor recurrence. Sixteen of 21 patients underwent microsurgical resection prior to IBT; in 5 patients, IBT was performed primarily after stereotactic biopsy for histological diagnosis. The cumulative tumor surface dose ranged from 50-65 Gy treating a median tumor volume of 3.6 ml (range, 0.3-11.6 ml). A median follow-up period of 105.3 months (range, 12.7-286.2 months) was evaluated. RESULTS: Actuarial 2-, 5- and 10-years overall- and disease-specific survival rates after IBT were each 90% and 100% at all times for ependymomas WHO I/II, for anaplastic ependymomas WHO III 100%, 100%, 70% and 100%, 100%, 86%, respectively. The neurological status of seven patients improved, while there was no change in 12 and deterioration in 2 patients, respectively. Follow-up MR images disclosed a complete tumor remission in 3, a partial remission in 12 and a stable disease in 6 patients. Treatment-associated morbidity only occurred in a single patient. CONCLUSIONS: This study shows that stereotactic IBT for intracranial ependymomas is safe and can provide a high degree of local tumor control. Due to the low rate of side effects, IBT may evolve into an attractive alternative to microsurgery in ependymomas located in eloquent areas or as a salvage treatment.

Precursor-directed syntheses and biological evaluation of new elansolid derivatives.

The antibiotic elansolid C1 (8) was isolated from Chitinophaga sancti strain FxGBF13 after fermentation in the presence of anthranilic acid. Remarkably, 8 was also obtained by addition of anthranilic acid to a crude fermentation extract containing the macrolide elansolid A2 (1*). This Michael-type conjugate addition allowed us to generate 21 new derivatives of elansolid C1 (9-29) by using various nucleophiles. Biological activities of all derivatives were evaluated against Staphylococcus aureus, Micrococcus luteus, and the mouse cell line L929.

The impact of bacterial genomics on natural product research.

"There's life in the old dog yet!" This adage also holds true for natural product research. After the era of natural products was declared to be over, because of the introduction of combinatorial synthesis techniques, natural product research has taken a surprising turn back towards a major field of pharmaceutical research. Current challenges, such as emerging multidrug-resistant bacteria, might be overcome by developments which combine genomic knowledge with applied biology and chemistry to identify, produce, and alter the structure of new lead compounds. Significant biological activity is reported much less frequently for synthetic compounds, a fact reflected in the large proportion of natural products and their derivatives in clinical use. This Review describes the impact of microbial genomics on natural products research, in particularly the search for new lead structures and their optimization. The limitations of this research are also discussed, thus allowing a look into future developments.

In vivo pro-apoptotic and antitumor efficacy of a c-Raf antisense phosphorothioate oligonucleotide: relationship to tumor size.

Previously, we have shown that a phosphorothioate antisense oligonucleotide (ODN) targeted against c-raf RNA (ISIS5132; cRaf-AS) induces apoptosis in human tumor cells. We now show that the same ODN also efficiently triggers apoptosis in human tumor xenografts in nu/nu mice. Although cRaf-AS showed a clearly inhibitory effect on the growth of established tumors (approximately 150 mm3) compared to a mismatched control ODN (MM), tumor progression was not prevented. This correlated with a partial refractoriness of the tumor to cRaf-AS-induced cell killing, which seemed to be due to an inhomogeneous and inefficient penetration of the ODN into the tumor tissue rather than cellular resistance. In agreement with this conclusion, we found that growth of small tumors (<50 mm3) was completely inhibited concomitantly with an accumulation of the ODN throughout the tumor. These data show that the cRaf-AS is a highly efficacious antitumor agent, provided accessibility into the tumor tissue is warranted, and suggest that PS-AS-ODN treatment may be particularly useful in an adjuvant setting.