Bacterial diversity and chemical ecology of natural product-producing bacteria from Great Salt Lake sediment.

Great Salt Lake (GSL), located northwest of Salt Lake City, UT, is the largest terminal lake in the USA. While the average salinity of seawater is ~3.3%, the salinity in GSL ranges between 5% and 28%. In addition to being a hypersaline environment, GSL also contains toxic concentrations of heavy metals, such as arsenic, mercury, and lead. The extreme environment of GSL makes it an intriguing subject of study, both for its unique microbiome and its potential to harbor novel natural product-producing bacteria, which could be used as resources for the discovery of biologically active compounds. Though work has been done to survey and catalog bacteria found in GSL, the Lake's microbiome is largely unexplored, and little to no work has been done to characterize the natural product potential of GSL microbes. Here, we investigate the bacterial diversity of two important regions within GSL, describe the first genomic characterization of Actinomycetota isolated from GSL sediment, including the identification of two new Actinomycetota species, and provide the first survey of the natural product potential of GSL bacteria.

Topical delivery of seriniquinone for treatment of skin cancer and fungal infections is enabled by a liquid crystalline lamellar phase.

Seriniquinone (SQ) was initially described by our group as an antimelanoma drug candidate and now also as an antifungal drug candidate. Despite its promising in vitro effects, SQ translation has been hindered by poor water-solubility. In this paper, we described the challenging nanoformulation process of SQ, which culminated in the selection of a phosphatidylcholine-based lamellar phase (PLP1). Liposomes and nanostructured lipid carriers were also evaluated but failed to encapsulate the compound. SQ-loaded PLP1 (PLP1-SQ) was characterized for the presence of sedimented or non-dissolved SQ, rheological and thermal behavior, and irritation potential with hen's egg test on the chorioallantoic membrane (HET-CAM). PLP1 influence on transepidermal water loss (TEWL) and skin penetration of SQ was assessed in a porcine ear skin model, while biological activity was evaluated against melanoma cell lines (SK-MEL-28 and SK-MEL-147) and C. albicans SC5314. Despite the presence of few particles of non-dissolved SQ (observed under the microscope 2 days after formulation obtainment), PLP1 tripled SQ retention in viable skin layers compared to SQ solution at 12 h. This effect did not seem to relate to formulation-induced changes on the barrier function, as no increases in TEWL were observed. No sign of vascular toxicity in the HET-CAM model was observed after cutaneous treatment with PLP1. SQ activity was maintained on melanoma cells after 48 h-treatment (IC50 values of 0.59-0.98 µM) whereas the minimum inhibitory concentration (MIC) against C. albicans after 24 h-treatment was 32-fold higher. These results suggest that a safe formulation for SQ topical administration was developed, enabling further in vivo studies.

Optimization of cancer immunotherapy on the basis of programmed death ligand-1 distribution and function.

Programmed cell death protein-1 (PD-1)/programmed death ligand-1 (PD-L1) immune checkpoint blockade as a breakthrough in cancer immunotherapy has shown unprecedented positive outcomes in the clinic. However, the overall effectiveness of PD-L1 antibody is less than expected. An increasing number of studies have demonstrated that PD-L1 is widely distributed and expressed not only on the cell membrane but also on the inside of the cells as well as on the extracellular vesicles secreted by tumour cells. Both endogenous and exogenous PD-L1 play significant roles in influencing the therapeutic effect of anti-tumour immunity. Herein, we mainly focused on the distribution and function of PD-L1 and further summarized the potential targeted therapeutic strategies. More importantly, in addition to taking the overall expression abundance of PD-L1 as a predictive indicator for selecting corresponding PD-1/PD-L1 monoclonal antibodies (mAbs), we also proposed that personalized combination therapies based on the different distribution of PD-L1 are worth attention to achieve more efficient and effective therapeutic outcomes in cancer patients. LINKED ARTICLES: This article is part of a themed issue on Cancer Microenvironment and Pharmacological Interventions. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v181.2/issuetoc.

Bacterial Diversity and Chemical Ecology of Natural Product-Producing Bacteria from Great Salt Lake Sediment.

Great Salt Lake (GSL), located northwest of Salt Lake City, UT, is the largest terminal lake in the United States. While the average salinity of seawater is ~3.3%, the salinity in GSL ranges between 5-28%. In addition to being a hypersaline environment, GSL also contains toxic concentrations of heavy metals, such as arsenic, mercury, and lead. The extreme environment of GSL makes it an intriguing subject of study, both for its unique microbiome and its potential to harbor novel natural product-producing bacteria, which could be used as resources for the discovery of biologically active compounds. Though work has been done to survey and catalogue bacteria found in GSL, the Lake's microbiome is largely unexplored, and little-to-no work has been done to characterize the natural product potential of GSL microbes. Here, we investigate the bacterial diversity of two important regions within GSL, describe the first genomic characterization of Actinomycetota isolated from GSL sediment, including the identification of a new Saccharomonospora species, and provide the first survey of the natural product potential of GSL bacteria.

High-Throughput Screen of Microbial Metabolites Identifies F1FO ATP Synthase Inhibitors as New Leads for Naegleria fowleri Infection.

Primary amebic meningoencephalitis (PAM), a brain infection caused by a free-living ameba Naegleria fowleri, leads to an extensive inflammation of the brain and death within 1-18 (median 5) days after symptoms begin. Although natural products have played a significant role in the development of drugs for over a century, research focusing on identifying new natural product-based anti-N. fowleri agents is limited. We undertook a large-scale ATP bioluminescence-based screen of about 10,000 unique marine microbial metabolite mixtures against the trophozoites of N. fowleri. Our screen identified about 100 test materials with >90% inhibition at 50 µg/mL and a dose-response study found 20 of these active test materials exhibiting an EC50 ranging from 0.2 to 2 µg/mL. Examination of four of these potent metabolite mixtures, derived from our actinomycete strains CNT671, CNT756, and CNH301, resulted in the isolation of a pure metabolite identified as oligomycin D. Oligomycin D exhibited nanomolar potency on multiple genotypes of N. fowleri, and it was five- or 850-times more potent than the recommended drugs amphotericin B or miltefosine. Oligomycin D is fast-acting and reached its EC50 in 10 h, and it was also able to inhibit the invasiveness of N. fowleri significantly when tested on a matrigel invasion assay. Since oligomycin is known to manifest inhibitory activity against F1FO ATP synthase, we tested different F1FO ATP synthase inhibitors and identified a natural peptide leucinostatin as a fast-acting amebicidal compound with nanomolar potency on multiple strains.

Actinoquinazolinone, a New Quinazolinone Derivative from a Marine Bacterium Streptomyces sp. CNQ-617, Suppresses the Motility of Gastric Cancer Cells.

A HPLC-UV guided fractionation of the culture broth of Streptomyces sp. CNQ-617 has led to the isolation of a new quinazolinone derivative, actinoquinazolinone (1), as well as two known compounds, 7-hydroxy-6-methoxy-3,4-dihydroquinazolin-4-one (2) and 7-methoxy-8-hydroxy cycloanthranilylproline (3). The interpretation of 1D, 2D NMR, and MS spectroscopic data revealed the planar structure of 1. Furthermore, compound 1 suppressed invasion ability by inhibiting epithelial-mesenchymal transition markers (EMT) in AGS cells at a concentration of 5 µM. In addition, compound 1 decreased the expression of seventeen genes related to human cell motility and slightly suppressed the signal transducer and activator of the transcription 3 (STAT3) signal pathway in AGS cells. Together, these results demonstrate that 1 is a potent inhibitor of gastric cancer cells.

Targeted and Logical Discovery of Piperazic Acid-Bearing Natural Products Based on Genomic and Spectroscopic Signatures.

A targeted and logical discovery method was devised for natural products containing piperazic acid (Piz), which is biosynthesized from ornithine by l-ornithine N-hydroxylase (KtzI) and N-N bond formation enzyme (KtzT). Genomic signature-based screening of a bacterial DNA library (2020 strains) using polymerase chain reaction (PCR) primers targeting ktzT identified 62 strains (3.1%). The PCR amplicons of KtzT-encoding genes were phylogenetically analyzed to classify the 23 clades into two monophyletic groups, I and II. Cultivating hit strains in media supplemented with 15NH4Cl and applying 1H-15N heteronuclear multiple bond correlation (HMBC) along with 1H-15N heteronuclear single quantum coherence (HSQC) and 1H-15N HSQC-total correlation spectroscopy (HSQC-TOCSY) NMR experiments detected the spectroscopic signatures of Piz and modified Piz. Chemical investigation of the hit strains prioritized by genomic and spectroscopic signatures led to the identification of a new azinothricin congener, polyoxyperuin B seco acid (1), previously reported chloptosin (2) in group I, depsidomycin D (3) incorporating two dehydropiperazic acids (Dpz), and lenziamides A and B (4 and 5), structurally novel 31-membered cyclic decapeptides in group II. By consolidating the phylogenetic and chemical analyses, clade-structure relationships were elucidated for 19 of the 23 clades. Lenziamide A (4) inhibited STAT3 activation and induced G2/M cell cycle arrest, apoptotic cell death, and tumor growth suppression in human colorectal cancer cells. Moreover, lenziamide A (4) resensitized 5-fluorouracil (5-FU) activity in both in vitro cell cultures and the in vivo 5-FU-resistant tumor xenograft mouse model. This work demonstrates that the genomic and spectroscopic signature-based searches provide an efficient and general strategy for new bioactive natural products containing specific structural motifs.

Nanoscaled Discovery of a Shunt Rifamycin from Salinispora arenicola Using a Three-Color GFP-Tagged Staphylococcus aureus Macrophage Infection Assay.

Antimicrobial resistance has emerged as a global public health threat, and development of novel therapeutics for treating infections caused by multi-drug resistant bacteria is urgent. Staphylococcus aureus is a major human and animal pathogen, responsible for high levels of morbidity and mortality worldwide. The intracellular survival of S. aureus in macrophages contributes to immune evasion, dissemination, and resilience to antibiotic treatment. Here, we present a confocal fluorescence imaging assay for monitoring macrophage infection by green fluorescent protein (GFP)-tagged S. aureus as a front-line tool to identify antibiotic leads. The assay was employed in combination with nanoscaled chemical analyses to facilitate the discovery of a new, active rifamycin analogue. Our findings indicate a promising new approach for the identification of antimicrobial compounds with macrophage intracellular activity. The antibiotic identified here may represent a useful addition to our armory in tackling the silent pandemic of antimicrobial resistance.

Expanding the Utility of Bioinformatic Data for the Full Stereostructural Assignments of Marinolides A and B, 24- and 26-Membered Macrolactones Produced by a Chemically Exceptional Marine-Derived Bacterium.

Marinolides A and B, two new 24- and 26-membered bacterial macrolactones, were isolated from the marine-derived actinobacterium AJS-327 and their stereostructures initially assigned by bioinformatic data analysis. Macrolactones typically possess complex stereochemistry, the assignments of which have been one of the most difficult undertakings in natural products chemistry, and in most cases, the use of X-ray diffraction methods and total synthesis have been the major methods of assigning their absolute configurations. More recently, however, it has become apparent that the integration of bioinformatic data is growing in utility to assign absolute configurations. Genome mining and bioinformatic analysis identified the 97 kb mld biosynthetic cluster harboring seven type I polyketide synthases. A detailed bioinformatic investigation of the ketoreductase and enoylreductase domains within the multimodular polyketide synthases, coupled with NMR and X-ray diffraction data, allowed for the absolute configurations of marinolides A and B to be determined. While using bioinformatics to assign the relative and absolute configurations of natural products has high potential, this method must be coupled with full NMR-based analysis to both confirm bioinformatic assignments as well as any additional modifications that occur during biosynthesis.

Marinobazzanan, a Bazzanane-Type Sesquiterpenoid, Suppresses the Cell Motility and Tumorigenesis in Cancer Cells.

Marinobazzanan (1), a new bazzanane-type sesquiterpenoid, was isolated from a marine-derived fungus belonging to the genus Acremonium. The chemical structure of 1 was elucidated using NMR and mass spectroscopic data, while the relative configurations were established through the analysis of NOESY data. The absolute configurations of 1 were determined by the modified Mosher's method as well as vibrational circular dichroism (VCD) spectra calculation and it was determined as 6R, 7R, 9R, and 10R. It was found that compound 1 was not cytotoxic to human cancer cells, including A549 (lung cancer), AGS (gastric cancer), and Caco-2 (colorectal cancer) below the concentration of 25 µM. However, compound 1 was shown to significantly decrease cancer-cell migration and invasion and soft-agar colony-formation ability at concentrations ranging from 1 to 5 µM by downregulating the expression level of KITENIN and upregulating the expression level of KAI1. Compound 1 suppressed ß-catenin-mediated TOPFLASH activity and its downstream targets in AGS, A549, and Caco-2 and slightly suppressed the Notch signal pathway in three cancer cells. Furthermore, 1 also reduced the number of metastatic nodules in an intraperitoneal xenograft mouse model.