Targeted Isolation of Antibiofilm Compounds from Halophytic Endophyte Bacillus velezensis 7NPB-3B Using LC-HR-MS-Based Metabolomics.

The discovery of new natural products has become more challenging because of the re-isolation of compounds and the lack of new sources. Microbes dwelling in extreme conditions of high salinity and temperature are huge prospects for interesting natural metabolites. In this study, the endophytic bacteria Bacillus velezensis 7NPB-3B isolated from the halophyte Salicornia brachiata was screened for its biofilm inhibition against methicillin-resistant Staphylococcus aureus (MRSA). The fractionation of the crude extract was guided by bioassay and LC-HRMS-based metabolomics using multivariate analysis. The 37 fractions obtained by high-throughput chromatography were dereplicated using an in-house MS-Excel macro coupled with the Dictionary of Natural Products database. Successive bioactivity-guided separation yielded one novel compound (1), a diketopiperazine (m/z 469.258 [M - H]-) with an attached saturated decanoic acid chain, and four known compounds (2-5). The compounds were identified based on 1D- and 2D-NMR and mass spectrometry. Compounds 1 and 5 exhibited excellent biofilm inhibition properties of >90% against the MRSA pathogen at minimum inhibition concentrations of 25 and 35 µg/mL, respectively. The investigation resulted in the isolation of a novel diketopiperazine from a bacterial endophyte of an untapped plant using an omics approach.

Bacillinaphthin A: A New Naphthohydroquinone from the Endophyte Bacillus subtilis NPROOT3.

The present study explores the endophyte associated with the halophyte Salicornia brachiata for uncovering new biologically important compounds. Thus, HPLC-PDA guided chemical investigation of the ethyl acetate extract of the Bacillus subtilis NPROOT3 led to the isolation of a new compound named bacillinaphthin A (1) along with previously known rubinaphthin A (2). The structure of 2 was determined by a comparison of HR-ESI-MS, 1 H and 13 C nuclear magnetic resonances (NMR) with those of reported data, whereas the structure of new compound 1 was elucidated by interpretation of 1D- and 2D-NMR and MS data. Bacillinaphthin (1) and rubinaphthin (2) feature 1,4-dihydroxy-2-naphthoic acid derivatives which have been isolated herein for the first time from the genus Bacillus. Bacillinaphthin (1) is a new congener of 2 with an additional succinic acid side chain attached to the sugar moiety. Production of succinoglycan compounds was reported to regulate symbiosis, hence the isolation of 1 exhibits an example of chemical ecology between the halophyte and its endophyte. In silico tools were used to assess the bioactive potential of both isolated molecules.

Dactylides A-C, three new bioactive 22-membered macrolides produced by Dactylosporangium aurantiacum.

Three new 22-membered polyol macrolides, dactylides A-C (1-3), were isolated from Dactylosporangium aurantiacum ATCC 23491 employing repeated chromatographic separations, and their structures were established based on detailed analysis of NMR and MS data. The relative configurations at the stereocenters were established via vicinal 1H-1H coupling constants, NOE correlations, and by application of Kishi's universal NMR database. In order to get insights into the biosynthetic pathway of 1-3, the genome sequence of the producer strain D. aurantiacum was obtained and the putative biosynthetic gene cluster encoding their biosynthesis was identified through bioinformatic analysis using antiSMASH. Compounds 1-3 showed significant in-vitro antimycobacterial and cytotoxic activity.

Bacillibactin class siderophores produced by the endophyte Bacillus subtilis NPROOT3 as antimycobacterial agents.

The bacterial endophytes isolated from the halophyte Salicornia brachiata were explored for the antimicrobial potential to discover novel microbial inhibitors that combat multidrug resistance. Upon investigation, ethyl acetate extract of the endophyte Bacillus subtilis NPROOT3 displayed significant potency against Mycobacterium smegmatis MTCC6 as well as Mycobacterium tuberculosis H37Rv strain. Further investigation of ethyl acetate crude extract by repeated chromatographic separations followed by characterization using UV, HR-ESI-MS, MALDI-MS, MALDI-MS/MS, CD, and NMR spectroscopy yielded a series of five known siderophores, namely, SVK21 (1), bacillibactin C (2), bacillibactin B (3), tribenglthin A (4), and bacillibactin (5). A total of two out of five compounds, 4 (MIC 38.66 µM) and 5 (MIC 22.15 µM) exhibited significant inhibition against the strain M. smegmatis MTCC6 comparable with positive control rifampicin (MIC 12.15 µM). None of these five bacillibactin molecules are previously reported to exhibit bioactivity against Mycobacterium sp. Herein for the first time, all the compounds were screened for their antibacterial activities against a panel of bacterial pathogens of humans. Furthermore, the probable mechanism of action of bacillibactin compounds for their antimycobacterial activity is also discussed. The findings of this study open up a new chemotype for inhibition of the Mycobacterium sp. and other multidrug-resistant pathogens.

Selective Partition of Lipopeptides from Fermentation Broth: A Green and Sustainable Approach.

Lipopeptide (LP) biosurfactants from microbes have the potential to gradually replace chemical synthetic surfactants and fit the contemporary green and sustainable industrial production concept. However, their active participation is comparatively low in the global market pertaining to their low yield in microbial broth and costly downstream processes arising due to tedious isolation and purification methods. Herein, an efficient extraction method is developed that utilizes an aqueous biphasic system (ABS) comprising ionic liquids and polypropylene glycol 400 (PPG) to selectively extract a mixture of cyclic lipopeptides, namely, surfactin and fengycin from the culture broth of Bacillus amyloliquefaciens 5NPA-1, isolated from the halophyte Salicornia brachiata Roxb. Out of four different ABSs, the ABS composed of 2-hydroxyethyl ammonium formate and PPG displayed a maximum extraction efficiency of 82.30%. PPG-rich phase containing lipopeptides exhibited excellent antimicrobial and mosquito larvicidal properties with no toxic effect on plants. The developed method is simple, novel and accelerates the application of cyclic lipopeptides produced by the microbial source.

Indazole-based microtubule-targeting agents as potential candidates for anticancer drugs discovery.

Tremendous research is focused on developing novel drug candidates targeting microtubules to inhibit their function in several cellular processes, including cell division. In this regard, several indazole derivatives were sought to target the colchicine binding site on the ß-tubulin, a crucial protein required to form microtubules, to develop microtubule targeting agents. Even though there are several reviews on the indazole-based compounds, none of them focused on using indazole scaffold to develop microtubule targeting agents. Therefore, this review aims to present the advances in research on compounds containing indazole scaffolds as microtubule targeting agents based on the articles published in the last two decades. Among the articles reviewed, we found that compounds 6 and 7 showed the lowest IC50 values of 0.6 âˆ¼ 0.9 nM in the cell line studies, making them the strongest indazole derivatives that target microtubules. The compounds 30, 31, 37 (IC50 = ∼ 1 nM) and compounds 8, 38 (IC50 = ∼ 2 nM) have proved to be potent microtubule inhibitors. The compounds 18, 31, 44, 45 also showed strong anticancer activity (IC50 = ∼ 8 nM). It is important to notice that except for compounds 9, 12, 13, 15, and SRF, the top activity compounds including 6, 7, 8, 10, 11, 30, 31, 37, 44, and 45 contain 3,4,5­trimethoxyphenyl substitution similar to that of colchicine. Therefore, it appears that the 3,4,5­trimethoxyphenyl substituent on the indazole scaffold is crucial for targeting CBS.

Synthesis and evaluation of 2-aryl-1H-benzo[d]imidazole derivatives as potential microtubule targeting agents.

Microtubule targeting agents (MTAs) are the potential drug candidates for anticancer drug discovery. Disrupting the microtubule formation or inhibiting the de-polymerization process by a synthetic molecule can lead to an excellent anticancer drug candidate. Here, we present the 2,5-substituted-1H-benzo[d]imidazole derivatives as potential colchicine, nocodazole binding site targeting agents. About 20 benzimidazole derivatives were synthesized with 82.0%-94.0% yield using mild reaction conditions. The synthesized compounds showed moderate to excellent anticancer activity established in three cell lines, including Hela cells, A549 cells, MRC-5 cells. The compounds B15, B16, B19, and B20 are the potential candidates with the IC50 values <15 µM in the three different cell lines. In MTT assay, compounds B15, B16, B19, and B20 showed excellent antiproliferation activity indicated by IC50 values in the range of 5.3 ± 0.21 to 18.1 ± 0.32 µM using HeLa and A549 cell lines. The predicted absorption, distribution, metabolism and excretion (ADME) properties and drug-likeness properties of B15, B16, B19, and B20 indicate that these compounds can be used as lead compounds for further study to develop excellent MTAs.

Development of a Novel Benzimidazole-Based Probe and Portable Fluorimeter for the Detection of Cysteine in Human Urine.

The measurement of cysteine in human urine and live cells is crucial for evaluating biological metabolism, monitoring and maintaining the immune system, preventing tissue/DNA damage caused by free radicals, preventing autoimmune diseases, and diagnosing disorders such as cystinuria and cancer. A method that uses a fluorescence turn-on probe and a portable fluorescence spectrometer device are crucial for highly sensitive, simple, rapid, and inexpensive cysteine detection. Herein, we present the synthesis and application of a benzimidazole-based fluorescent probe (ABIA) along with the design and development of a portable fluorescence spectrometer device (CysDDev) for detecting cysteine in simulated human urine. ABIA showed excellent selectivity and sensitivity in detecting cysteine over homocysteine, glutathione, and other amino acids with the response time of 1 min and demonstrated a detection limit of 16.3 nM using the developed CysDDev. Further, ABIA also demonstrated its utility in detecting intracellular cysteine, making it an excellent probe for bio-imaging assay.

Microbial melanin: Recent advances in biosynthesis, extraction, characterization, and applications.

Melanin is a common name for a group of biopolymers with the dominance of potential applications in medical sciences, cosmeceutical, bioremediation, and bioelectronic applications. The broad distribution of these pigments suggests their role to combat abiotic and biotic stresses in diverse life forms. Biosynthesis of melanin in fungi and bacteria occurs by oxidative polymerization of phenolic compounds predominantly by two pathways, 1,8-dihydroxynaphthalene [DHN] or 3,4-dihydroxyphenylalanine [DOPA], resulting in different kinds of melanin, i.e., eumelanin, pheomelanin, allomelanin, pyomelanin, and neuromelanin. The enzymes responsible for melanin synthesis belong mainly to tyrosinase, laccase, and polyketide synthase families. Studies have shown that manipulating culture parameters, combined with recombinant technology, can increase melanin yield for large-scale production. Despite significant efforts, its low solubility has limited the development of extraction procedures, and heterogeneous structural complexity has impaired structural elucidation, restricting effective exploitation of their biotechnological potential. Innumerable studies have been performed on melanin pigments from different taxa of life in order to advance the knowledge about melanin pigments for their efficient utilization in diverse applications. These studies prompted an urgent need for a comprehensive review on melanin pigments isolated from microorganisms, so that such review encompassing biosynthesis, bioproduction, characterization, and potential applications would help researchers from diverse background to understand the importance of microbial melanins and to utilize the information from the review for planning studies on melanin. With this aim in mind, the present report compares conventional and modern ideas for environment-friendly extraction procedures for melanin. Furthermore, the characteristic parameters to differentiate between eumelanin and pheomelanin are also mentioned, followed by their biotechnological applications forming the basis of industrial utilization. There lies a massive scope of work to circumvent the bottlenecks in their isolation and structural elucidation methodologies.

Biodiversity and antimicrobial potential of bacterial endophytes from halophyte Salicornia brachiata.

Extreme natural habitats like halophytes, marsh land, and marine environment are suitable arena for chemical ecology between plants and microbes having environmental impact. Endophytes are an ecofriendly option for the promotion of plant growth and to serve as sustainable resource of novel bioactive natural products. The present study, focusing on biodiversity of bacterial endophytes from Salicornia brachiata, led to isolation of around 336 bacterial endophytes. Phylogenetic analysis of 63 endophytes revealed 13 genera with 27 different species, belonging to 3 major groups: Firmicutes, Proteobacteria, and Actinobacteria. 30% endophytic isolates belonging to various genera demonstrated broad-spectrum antibacterial and antifungal activities against a panel of human, plant, and aquatic infectious agents. An endophytic isolate Bacillus amyloliquefaciens 5NPA-1, exhibited strong in-vitro antibacterial activity against human pathogen Staphylococcus aureus and phytopathogen Xanthomonas campestris. Investigation through LC-MS/MS-based molecular networking and bioactivity-guided purification led to the identification of three bioactive compounds belonging to lipopeptide class based on 1H-, 13C-NMR and MS analysis. To our knowledge, this is the first report studying bacterial endophytic biodiversity of Salicornia brachiata and the isolation of bioactive compounds from its endophyte. Overall, the present study provides insights into the diversity of endophytes associated with the plants from the extreme environment as a rich source of metabolites with remarkable agricultural applications and therapeutic properties.

Macrocycles and Supramolecules as Antioxidants: Excellent Scaffolds for Development of Potential Therapeutic Agents.

Oxidative stress due to the high levels of reactive oxygen species (ROS) that damage biomolecules (lipids, proteins, DNA) results in acute inflammation. However, without proper intervention, acute inflammation progresses to chronic inflammation and then to several chronic diseases, including cancer, myocardial infarction, cardiovascular diseases, chronic inflammation, atherosclerosis, and more. There has been extensive research on the antioxidants of natural origin. However, there are myriad possibilities for the development of synthetic antioxidants for pharmacological applications. There is an increasing interest in the identification of novel synthetic antioxidants for the modulation of biochemical processes related to ROS. In this regard, derivatives of supramolecules, such as calix[n]arene, resorcinarene, calixtyrosol, calixpyrrole, cucurbit[n]uril, porphyrin etc. are gaining attention for their abilities to scavenge the free radicals. Supramolecular chemistry offers excellent scaffolds for the development of novel antioxidants that can be used to modulate free radical reactions and to improve the disorders related to oxidative stress. This review focuses on the interdisciplinary approach for the design and development of novel synthetic antioxidants based on supramolecular scaffolds, with potentially protective effects against oxidative stress.

Natural Melanin Produced by the Endophytic Bacillus subtilis 4NP-BL Associated with the Halophyte Salicornia brachiata.

Natural melanin with many interesting properties has potential applications in cosmetics, drug delivery, semiconductors, etc. However, conventional production methods are not efficient, resulting in its high cost (350-650 USD g-1), which has been a bottleneck for its efficient commercial utilization. To explore a faster extraction method with a higher yield, a melanin-producing endophytic bacterium was isolated from the halophyte Salicornia brachiata and further identified as Bacillus subtilis 4NP-BL by phylogenetic analysis of 16S rRNA gene sequences. The maximum melanin yield of up to 1.5 g dry wt L-1 of production media was obtained through central composite design (CCD). The isolated melanin belonged to the eumelanin class with an irregular structure on the basis of elemental analysis, UV-vis, Fourier transform infrared (FT-IR), scanning electron microscopy (SEM), electron paramagnetic resonance (EPR), and NMR studies. Furthermore, purified melanin displayed antioxidant activity and antimicrobial activity against pathogens Xanthomonas campestris and Alteromonas macleodii. Thus, this study further suggests a probable role of endophytes that produce melanin in aiding host plant protection from environmental stress and other pathogens.

Combinatorial biosynthesis and antibacterial evaluation of glycosylated derivatives of 12-membered macrolide antibiotic YC-17.

Expression plasmids carrying different deoxysugar biosynthetic gene cassettes and the gene encoding a substrate-flexible glycosyltransferase DesVII were constructed and introduced into Streptomyces venezuelae YJ003 mutant strain bearing a deletion of a desosamine biosynthetic (des) gene cluster. The resulting recombinants produced macrolide antibiotic YC-17 analogs possessing unnatural sugars replacing native D-desosamine. These metabolites were isolated and further purified using chromatographic techniques and their structures were determined as D-quinovosyl-10-deoxymethynolide, L-rhamnosyl-10-deoxymethynolide, L-olivosyl-10-deoxymethynolide, and D-boivinosyl-10-deoxymethynolide on the basis of 1D and 2D NMR and MS analyses and the stereochemistry of sugars was confirmed using coupling constant values and NOE correlations. Their antibacterial activities were evaluated in vitro against erythromycin-susceptible and -resistant Enterococcus faecium and Staphylococcus aureus. Substitution with L-rhamnose displayed better antibacterial activity than parent compound YC-17 containing native sugar D-desosamine. The present study on relationships between chemical structures and antibacterial activities could be useful in generation of novel advanced antibiotics utilizing combinatorial biosynthesis approach.

Characterization of FK506 biosynthetic intermediates involved in post-PKS elaboration.

The post-PKS modification steps of FK506 biosynthesis include C9-oxidation and 31-O-methylation, but the sequence of these reactions and the exact route have remained unclear. This study details the post-PKS modification pathways in FK506 biosynthesis through the identification of all intermediates and in vitro enzymatic reactions of the cytochrome P450 hydroxylase FkbD and the methyltransferase FkbM. These results complete our understanding of post-PKS modification steps to FK506 showing the substrate flexibility of two enzymes involved and the existence of two parallel biosynthetic routes to FK506.

Engineered biosynthesis of glycosylated derivatives of narbomycin and evaluation of their antibacterial activities.

A 14-membered macrolide antibiotic narbomycin produced from Streptomyces venezuelae ATCC 15439 is composed of polyketide macrolactone ring and D-desosamine as a deoxysugar moiety, which acts as an important determinant of its antibacterial activity. In order to generate diverse glycosylated derivatives of narbomycin, expression plasmids carrying different deoxysugar biosynthetic gene cassettes and the gene encoding a substrate-flexible glycosyltransferase DesVII were constructed and introduced into S. venezuelae YJ003 mutant strain bearing a deletion of thymidine-5'-diphospho-D-desosamine biosynthetic gene cluster. The resulting recombinants of S. venezuelae produced a range of new analogs of narbomycin, which possess unnatural sugar moieties instead of native deoxysugar D-desosamine. The structures of narbomycin derivatives were determined through nuclear magnetic resonance spectroscopy and mass spectrometry analyses and their antibacterial activities were evaluated in vitro against erythromycin-susceptible and -resistant Enterococcus faecium and Staphylococcus aureus. Substitution with L-rhamnose or 3-O-demethyl-D-chalcose was demonstrated to exhibit greater antibacterial activity than narbomycin and the clinically relevant erythromycin. This work provides new insight into the functions of deoxysugar biosynthetic enzymes and structure-activity relationships of the sugar moieties attached to the macrolides and demonstrate the potential of combinatorial biosynthesis for the generation of new macrolides carrying diverse sugars with increased antibacterial activities.

Bile acid derivatives from a sponge-associated bacterium Psychrobacter sp.

In our search for bioactive metabolites from a marine sponge-associated bacterium Psychrobacter sp., a new bile acid derivative (1), which was assumed to be an artifact, were isolated along with six known (2-7) compounds by bioactivity-guided fractionation. Elucidation of the structure of the new compound was done using a combination of NMR ((1)H, (13)C, HMBC, HSQC, and COSY) and MS spectroscopy. Compound 1 exhibited moderate suppressive effects on both NO and IL-6 production at a concentration of 200 microM (87.3 microg/mL) without significant cytotoxicity against cells. Compounds 2-5 and 7 showed selective inhibitory activity against several human pathogenic bacterial strains at the low concentration of 30 microg/well. In a cytotoxicity evaluation, only compound 7 showed mild cytotoxicity against five human solid tumor cell lines (A-549, SK-OV-3, SK-MEL-2, XF-498, and HCT-15) with ED(50) values in the range of 11-14 microg/mL.

Cytotoxic bromotyrosine derivatives from a two-sponge association of Jaspis sp. and Poecillastra sp.

Bioassay-guided chemical investigation of the lipophilic extract of a two-sponge association (Jaspis sp. and Poecillastra sp.) led to the isolation of two new bromotyrosine derivatives (1 and 2), along with known derivatives (3-12). Cyclobispsammaplin A (1) is a cyclic derivative of the previously reported bispsammaplin A (13), while psammaplin M (2) is composed of beta-alanine (or aspartic acid) unit. Compounds 3, 4, 6, 10, and 12 are isolated for the first time from a sponge belonging to the subclass Tetractinomorpha. Structure elucidation was performed by a combination of high resolution mass and 2D NMR (principally COSY, HMBC, HSQC, and NOESY) spectroscopy. Compounds 1-4, 6, 10, and 12 were evaluated for cytotoxicity against a small panel of five human solid tumor cell lines and their activity was compared in relevance to their structure.

Furan metabolites from the sponge-derived yeast Pichia membranifaciens.

Five new compounds, pichiafurans A-C (1-3) and pichiacins A and B (4 and 5), along with five known compounds (6-10), have been isolated from the yeast Pichia membranifaciens derived from a marine sponge Petrosia sp. Their structures were elucidated by 1D and 2D NMR and mass spectrometry techniques. Pichiafurans are rare examples of monofurano metabolites isolated from yeast.

Anti-inflammatory constituents of the red alga Gracilaria verrucosa and their synthetic analogues.

A chemical study on the anti-inflammatory components of the red alga Gracilaria verrucosa led to the isolation of new 11-deoxyprostaglandins ( 1- 4), a ceramide ( 5), and a C 16 keto fatty acid ( 6), along with known oxygenated fatty acids ( 7- 14). Their structures were elucidated on the basis of NMR and MS data. The absolute configurations of compounds 1- 5 were determined by Mosher's method. The anti-inflammatory activity of the isolated compounds ( 1- 14) was evaluated by determining their inhibitory effects on the production of pro-inflammatory mediators (NO, IL-6, and TNF-alpha) in lipopolysaccharide (LPS)-activated RAW 264.7 murine macrophage cells. Compounds 9 and 10 exhibited the most potent activity. In the evaluation of these two compounds and derivatized analogues ( 15- 40), the anti-inflammatory activity was enhanced in some synthetic analogues. These enone fatty acids were investigated as potential anti-inflammatory leads for the first time.

Renierosides, cerebrosides from a marine sponge Haliclona (Reniera) sp.

Guided by the brine shrimp lethality assay, eight new cerebrosides (1-8) have been isolated from an extract of the marine sponge Haliclona (Reniera) sp. A novel feature of these cerebrosides was the presence of unprecedented amide-linked long-chain fatty acid moieties. The planar structures of the cerebrosides (1-8) were established by 1D and 2D NMR spectroscopic techniques, mass spectrometric analyses, and chemical degradation methods. The isolated compounds did not display cytotoxicity to a panel of five human solid tumor cell lines.