Marine natural products.

Covering: January to the end of December 2022This review covers the literature published in 2022 for marine natural products (MNPs), with 645 citations (633 for the period January to December 2022) referring to compounds isolated from marine microorganisms and phytoplankton, green, brown and red algae, sponges, cnidarians, bryozoans, molluscs, tunicates, echinoderms, the submerged parts of mangroves and other intertidal plants. The emphasis is on new compounds (1417 in 384 papers for 2022), together with the relevant biological activities, source organisms and country of origin. Pertinent reviews, biosynthetic studies, first syntheses, and syntheses that led to the revision of structures or stereochemistries, have been included. An analysis of NP structure class diversity in relation to biota source and biome is discussed.

Formation of Trideuteromethylated Artifacts of Pyrrole-Containing Natural Products.

Pyrrole-containing natural products form a large group of structurally diverse compounds that occur in both terrestrial and marine organisms. In the present study the formation of trideuteromethylated artifacts of pyrrole-containing natural products was investigated, focusing on the discorhabdins. Three deuterated discorhabdins, 1, 3, and 5, were identified to be isolation procedure artifacts caused by the presence of DMSO-d6 during NMR sample preparation and handling. Three additional semisynthetic derivatives, 7-9, were made during the investigation of the mechanism of formation, which was shown to be driven by trideuteromethyl radicals in the presence of water, methanol, TFA, and traces of iron in the deuterated solvent. Generation of trideuteromethylated artifacts was also confirmed for other classes of pyrrole-containing metabolites, namely, makaluvamines, tambjamines, and dibromotryptamines, which had also been dissolved in DMSO-d6 during the structure elucidation process. Semisynthetic discorhabdins were assessed for antiproliferative activity against a panel of human tumor cell lines, and 14-trideuteromethyldiscorhabdin L (3) averaged low micromolar potency.

An Investigation of Structure-Activity Relationships and Cell Death Mechanisms of the Marine Alkaloids Discorhabdins in Merkel Cell Carcinoma Cells.

A library of naturally occurring and semi-synthetic discorhabdins was assessed for their effects on Merkel cell carcinoma (MCC) cell viability. The set included five new natural products and semi-synthetic compounds whose structures were elucidated with NMR, HRMS, and ECD techniques. Several discorhabdins averaged sub-micromolar potency against the MCC cell lines tested and most of the active compounds showed selectivity towards virus-positive MCC cell lines. An investigation of structure-activity relationships resulted in an expanded understanding of the crucial structural features of the discorhabdin scaffold. Mechanistic cell death assays suggested that discorhabdins, unlike many other MCC-active small molecules, do not induce apoptosis, as shown by the lack of caspase activation, annexin V staining, and response to caspase inhibition. Similarly, discorhabdin treatment failed to increase MCC intracellular calcium and ROS levels. In contrast, the rapid loss of cellular reducing potential and mitochondrial membrane potential suggested that discorhabdins induce mitochondrial dysfunction leading to non-apoptotic cell death.

Screen for New Antimicrobial Natural Products from the NCI Program for Natural Product Discovery Prefractionated Extract Library.

The continuing emergence of antibiotic-resistant microbes highlights the need for the identification of new chemotypes with antimicrobial activity. One of the most prolific sources of antimicrobial molecules has been the systematic screening of natural product samples. The National Institute of Allergy and Infectious Diseases and the National Cancer Institute here report a large screen of 326,656 partially purified natural product fractions against a panel of four microbial pathogens, resulting in the identification of >3000 fractions with antifungal and/or antibacterial activity. A small sample of these active fractions was further purified and the chemical structures responsible for the antimicrobial activity were elucidated. The proof-of-concept study identified many different chemotypes, several of which have not previously been reported to have antimicrobial activity. The results show that there remain many unidentified antibiotic compounds from nature.

Biochemical Discovery, Intracellular Evaluation, and Crystallographic Characterization of Synthetic and Natural Product Adenosine 3',5'-Cyclic Monophosphate-Dependent Protein Kinase A (PKA) Inhibitors.

The recent demonstration that adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase A (PKA) plays an oncogenic role in a number of important cancers has led to a renaissance in drug development interest targeting this kinase. We therefore have established a suite of biochemical, cell-based, and structural biology assays for identifying and evaluating new pharmacophores for PKA inhibition. This discovery process started with a 384-well high-throughput screen of more than 200,000 substances, including fractionated natural product extracts. Identified active compounds were further prioritized in biochemical, biophysical, and cell-based assays. Priority lead compounds were assessed in detail to fully characterize several previously unrecognized PKA pharmacophores including the generation of new X-ray crystallography structures demonstrating unique interactions between PKA and bound inhibitor molecules.

National Cancer Institute (NCI) Program for Natural Product Discovery: Exploring NCI-60 Screening Data of Natural Product Samples with Artificial Neural Networks.

National Cancer Institute (NCI) Program for Natural Product Discovery is a new initiative aimed at creating new technologies for natural product-based drug discovery. Here, we present the development of a neural network-based bioinformatics platform for visualization and analysis of natural product high-throughput screening data using the NCI's 60 human tumor cell anticancer drug screen. We demonstrate how the tool enables visualization of similar patterns of response that can be parsed both chemically and taxonomically, grouping NCI-60 biological profiles in one easy-to-use bioinformatics interface.

Photochemical Dimerization of Plakinidine B Leads to Potent Inhibition of the E3 Ubiquitin-Protein Ligase CBL-B.

A new dimeric alkaloid plakoramine A [(±)-1] was identified from a marine sponge Plakortis sp. Chiral-phase HPLC separation of (±)-1 led to the purified enantiomers (+)-1 and (-)-1 which both potently inhibited CBL-B E3 ubiquitin ligase activities. The absolute configurations of the enantiomers were determined by quantum chemical calculations. Scrutinization of the purification conditions revealed a previously undescribed, nonenzymatic route to form (±)-1 via photochemical conversion of its naturally occurring monomeric counterpart, plakinidine B (2).

Molecular genomic features associated with in vitro response of the NCI-60 cancer cell line panel to natural products.

Natural products remain a significant source of anticancer chemotherapeutics. The search for targeted drugs for cancer treatment includes consideration of natural products, which may provide new opportunities for antitumor cytotoxicity as single agents or in combination therapy. We examined the association of molecular genomic features in the well-characterized NCI-60 cancer cell line panel with in vitro response to treatment with 1302 small molecules which included natural products, semisynthetic natural product derivatives, and synthetic compounds based on a natural product pharmacophore from the Developmental Therapeutics Program of the US National Cancer Institute's database. These compounds were obtained from a variety of plant, marine, and microbial species. Molecular information utilized for the analysis included expression measures for 23059 annotated transcripts, lncRNAs, and miRNAs, and data on protein-changing single nucleotide variants in 211 cancer-related genes. We found associations of expression of multiple genes including SLFN11, CYP2J2, EPHX1, GPC1, ELF3, and MGMT involved in DNA damage repair, NOTCH family members, ABC and SLC transporters, and both mutations in tyrosine kinases and BRAF V600E with NCI-60 responses to specific categories of natural products. Hierarchical clustering identified groups of natural products, which correlated with a specific mechanism of action. Specifically, several natural product clusters were associated with SLFN11 gene expression, suggesting that potential action of these compounds may involve DNA damage. The associations between gene expression or genome alterations of functionally relevant genes with the response of cancer cells to natural products provide new information about potential mechanisms of action of these identified clusters of compounds with potentially similar biological effects. This information will assist in future drug discovery and in design of new targeted cancer chemotherapy agents.

Triple-Negative Breast Cancer Cells Exhibit Differential Sensitivity to Cardenolides from Calotropis gigantea.

Triple-negative breast cancers (TNBC) are aggressive and heterogeneous cancers that lack targeted therapies. We implemented a screening program to identify new leads for subgroups of TNBC using diverse cell lines with different molecular drivers. Through this program, we identified an extract from Calotropis gigantea that caused selective cytotoxicity in BT-549 cells as compared to four other TNBC cell lines. Bioassay-guided fractionation of the BT-549 selective extract yielded nine cardenolides responsible for the selective activity. These included eight known cardenolides and a new cardenolide glycoside. Structure-activity relationships among the cardenolides demonstrated a correlation between their relative potencies toward BT-549 cells and Na+/K+ ATPase inhibition. Calotropin, the compound with the highest degree of selectivity for BT-549 cells, increased intracellular Ca2+ in sensitive cells to a greater extent than in the resistant MDA-MB-231 cells. Further studies identified a second TNBC cell line, Hs578T, that is also highly sensitive to the cardenolides, and mechanistic studies were conducted to identify commonalities among the sensitive cell lines. Experiments showed that both cardenolide-sensitive cell lines expressed higher mRNA levels of the Na+/Ca2+ exchanger NCX1 than resistant TNBC cells. This suggests that NCX1 could be a biomarker to identify TNBC patients that might benefit from the clinical administration of a cardiac glycoside for anticancer indications.

National Cancer Institute (NCI) Program for Natural Products Discovery: Rapid Isolation and Identification of Biologically Active Natural Products from the NCI Prefractionated Library.

An automated, high-capacity, and high-throughput procedure for the rapid isolation and identification of biologically active natural products from a prefractionated library is presented. The semipreparative HPLC method uses 1 mg of the primary hit fraction and produces 22 subfractions in an assay-ready format. Following screening, all active fractions are analyzed by NMR, LCMS, and FTIR, and the active principle structural classes are elucidated. In the proof-of-concept study, we show the processes involved in generating the subfractions, the throughput of the structural elucidation work, as well as the ability to rapidly isolate and identify new and biologically active natural products. Overall, the rapid second-stage purification conserves extract mass, requires much less chemist time, and introduces knowledge of structure early in the isolation workflow.

Creating and screening natural product libraries.

Covering: up to 2020The National Cancer Institute of the United States (NCI) has initiated a Cancer Moonshot program entitled the NCI Program for Natural Product Discovery. As part of this effort, the NCI is producing a library of 1 000 000 partially purified natural product fractions which are being plated into 384-well plates and provided to the research community free of charge. As the first 326 000 of these fractions have now been made available, this review seeks to describe the general methods used to collect organisms, extract those organisms, and create a prefractionated library. Importantly, this review also details both cell-based and cell-free bioassay methods and the adaptations necessary to those methods to productively screen natural product libraries. Finally, this review briefly describes post-screen dereplication and compound purification and scale up procedures which can efficiently identify active compounds and produce sufficient quantities of natural products for further pre-clinical development.

Using the Cancer Dependency Map to Identify the Mechanism of Action of a Cytotoxic Alkenyl Derivative from the Fruit of Choerospondias axillaris.

An extract prepared from the fruit of Choerospondias axillaris exhibited differential cytotoxic effects when tested in a panel of pediatric cancer cell lines [Ewing sarcoma (A-673), rhabdomyosarcoma (SJCRH30), medulloblastoma (D283), and hepatoblastoma (Hep293TT)]. Bioassay-guided fractionation led to the purification of five new hydroquinone-based metabolites, choerosponols A-E (1-5), bearing unsaturated hydrocarbon chains. The structures of the natural products were determined using a combination of 1D and 2D NMR, HRESIMS, ECD spectroscopy, and Mosher ester analyses. The purified compounds were evaluated for their antiproliferative and cytotoxic activities, revealing that 1, which contains a benzofuran moiety, exhibited over 50-fold selective antiproliferative activity against Ewing sarcoma and medulloblastoma cells with growth inhibitory (GI50) values of 0.19 and 0.07 µM, respectively. The effects of 1 were evaluated in a larger panel of cancer cell lines, and these data were used in turn to interrogate the Project Achilles cancer dependency database, leading to the identification of the MCT1 transporter as a functional target of 1. These data highlight the utility of publicly available cancer dependency databases such as Project Achilles to facilitate the identification of the mechanisms of action of compounds with selective activities among cancer cell lines, which can be a major challenge in natural products drug discovery.

The NCI library of traditional Chinese medicinal plant extracts - Preliminary assessment of the NCI-60 activity and chemical profiling of selected species.

Botanical-based natural products are an important resource for medicinal drug discovery and continue to provide diverse pharmacophores with therapeutic potential against cancer and other human diseases. A prototype Traditional Chinese Medicine (TCM) plant extract library has been established at the US National Cancer Institute, which contains both the organic and aqueous extracts of 132 authenticated medicinal plant species that collectively represent the potential therapeutic contents of most commonly used TCM herbal prescriptions. This library is publicly available in 96- and 384- well plates for high throughput screening across a broad array of biological targets, as well as in larger quantities for isolation of active chemical ingredients. Herein, we present the methodology used to generate the library and the preliminary assessment of the anti-proliferative activity of this crude extract library in NCI-60 human cancer cell lines screen. Particularly, we report the chemical profiling and metabolome comparison analysis of four commonly used TCM plants, namely Brucea javanica, Dioscorea nipponica, Cynanchum atratum, and Salvia miltiorrhiza. Bioassay-guided isolation resulted in the identification of the active compounds, and different extraction methods were compared for their abilities to extract cytotoxic compounds and to concentrate biologically active natural products.

Anacolosins A-F and Corymbulosins X and Y, Clerodane Diterpenes from Anacolosa clarkii Exhibiting Cytotoxicity toward Pediatric Cancer Cell Lines.

An extract of the plant Anacolosa clarkii was obtained from the NCI Natural Products Repository, and it showed cytotoxic activity toward several types of pediatric solid tumor cell lines. Bioassay-guided fractionation led to the purification of eight new clerodane diterpenes [anacolosins A-F (1-6) and corymbulosins X and Y (7 and 8)] and two known compounds (9 and 10) that contained an isozuelanin skeleton. The structures of the new natural products were determined using 1D and 2D NMR and HRESIMS data, while the relative and absolute configurations of the compounds were assessed using a combination of 1H NMR coupling constant data, ROESY experiments, ECD (electronic circular dichroism) and VCD (vibrational circular dichroism) spectroscopy, chemical methods (including Mosher and 2-naphthacyl esterification), and chiral HPLC analyses. The purified natural products exhibited a range of cytotoxic activities against cell lines representing four pediatric cancer types (i.e., rhabdomyosarcoma, Ewing sarcoma, medulloblastoma, and hepatoblastoma) with total growth inhibitory (TGI) values in the range 0.2-4.1 µM. The rhabdomyosarcoma and medulloblastoma cell lines showed higher sensitivity to compounds 1-4, which are the first compounds reported to contain an isozuelanin skeleton and feature keto carbonyl groups at the C-6 positions. In contrast, the hepatoblastoma cell line was modestly more sensitive to 7-10, which contained a C-6 hydroxy group moiety.

Erythrofordins D and E, two new cassaine-type diterpenes from Erythrophleum suaveolens.

Two new cassaine-type diterpenoids, namely erythrofordins D (1) and E (2), sourced from a Cameroon collection of Erythrophleum suaveolens were isolated and assessed for anti-tumor activity. In the NCI-60 cancer cell assay, erythrofordins D (1) and E (2) were found to be cytotoxic in the low micro molar ranges with a mean GI50 value of 2.45 and 0.71 µM, mean TGI value of 9.77 and 2.29 µM, and a mean LC50 of 26.92 and 11.48 µM for 1 and 2 respectively. Using the COMPARE algorithm, the new compounds were found to have similar NCI-60 response profiles to the known cardiac glycosides hyrcanoside and strophanthin. In addition, in an assay examining the viability and contractile function in human cardiomyocytes derived from induced pluripotent stem-cells, erythrofordins showed cardiotoxicity effects at concentrations as low as 0.03 µg/mL.

NCI Program for Natural Product Discovery: A Publicly-Accessible Library of Natural Product Fractions for High-Throughput Screening.

The US National Cancer Institute's (NCI) Natural Product Repository is one of the world's largest, most diverse collections of natural products containing over 230,000 unique extracts derived from plant, marine, and microbial organisms that have been collected from biodiverse regions throughout the world. Importantly, this national resource is available to the research community for the screening of extracts and the isolation of bioactive natural products. However, despite the success of natural products in drug discovery, compatibility issues that make extracts challenging for liquid handling systems, extended timelines that complicate natural product-based drug discovery efforts and the presence of pan-assay interfering compounds have reduced enthusiasm for the high-throughput screening (HTS) of crude natural product extract libraries in targeted assay systems. To address these limitations, the NCI Program for Natural Product Discovery (NPNPD), a newly launched, national program to advance natural product discovery technologies and facilitate the discovery of structurally defined, validated lead molecules ready for translation will create a prefractionated library from over 125,000 natural product extracts with the aim of producing a publicly-accessible, HTS-amenable library of >1,000,000 fractions. This library, representing perhaps the largest accumulation of natural-product based fractions in the world, will be made available free of charge in 384-well plates for screening against all disease states in an effort to reinvigorate natural product-based drug discovery.

HSQC-TOCSY Fingerprinting for Prioritization of Polyketide- and Peptide-Producing Microbial Isolates.

Microbial products are a promising source for drug leads as a result of their unique structural diversity. However, reisolation of already known natural products significantly hampers the discovery process, and it is therefore important to incorporate effective microbial isolate selection and dereplication protocols early in microbial natural product studies. We have developed a systematic approach for prioritization of microbial isolates for natural product discovery based on heteronuclear single-quantum correlation-total correlation spectroscopy (HSQC-TOCSY) nuclear magnetic resonance profiles in combination with antiplasmodial activity of extracts. The HSQC-TOCSY experiments allowed for unfractionated microbial extracts containing polyketide and peptidic natural products to be rapidly identified. Here, we highlight how this approach was used to prioritize extracts derived from a library of 119 ascidian-associated actinomycetes that possess a higher potential to produce bioactive polyketides and peptides.

Actinomycete Metabolome Induction/Suppression with N-Acetylglucosamine.

The metabolite profiles of three sponge-derived actinomycetes, namely, Micromonospora sp. RV43, Rhodococcus sp. RV157, and Actinokineospora sp. EG49 were investigated after elicitation with N-acetyl-d-glucosamine. 1H NMR fingerprint methodology was utilized to study the differences in the metabolic profiles of the bacterial extracts before and after elicitation. Our study found that the addition of N-acetyl-d-glucosamine modified the secondary metabolite profiles of the three investigated actinomycete isolates. N-Acetyl-d-glucosamine induced the production of 3-formylindole (11) and guaymasol (12) in Micromonospora sp. RV43, the siderophore bacillibactin 16, and surfactin antibiotic 17 in Rhodococcus sp. RV157 and increased the production of minor metabolites actinosporins E-H (21-24) in Actinokineospora sp. EG49. These results highlight the use of NMR fingerprinting to detect changes in metabolism following addition of N-acetyl-d-glucosamine. N-Acetyl-d-glucosamine was shown to have multiple effects including suppression of metabolites, induction of new metabolites, and increased production of minor compounds.

Potential of marine natural products against drug-resistant fungal, viral, and parasitic infections.

Antibiotics have revolutionised medicine in many aspects, and their discovery is considered a turning point in human history. However, the most serious consequence of the use of antibiotics is the concomitant development of resistance against them. The marine environment has proven to be a very rich source of diverse natural products with significant antibacterial, antifungal, antiviral, antiparasitic, antitumour, anti-inflammatory, antioxidant, and immunomodulatory activities. Many marine natural products (MNPs)-for example, neoechinulin B-have been found to be promising drug candidates to alleviate the mortality and morbidity rates caused by drug-resistant infections, and several MNP-based anti-infectives have already entered phase 1, 2, and 3 clinical trials, with six approved for usage by the US Food and Drug Administration and one by the EU. In this Review, we discuss the diversity of marine natural products that have shown in-vivo efficacy or in-vitro potential against drug-resistant infections of fungal, viral, and parasitic origin, and describe their mechanism of action. We highlight the drug-like physicochemical properties of the reported natural products that have bioactivity against drug-resistant pathogens in order to assess their drug potential. Difficulty in isolation and purification procedures, toxicity associated with the active compound, ecological impacts on natural environment, and insufficient investments by pharmaceutical companies are some of the clear reasons behind market failures and a poor pipeline of MNPs available to date. However, the diverse abundance of natural products in the marine environment could serve as a ray of light for the therapy of drug-resistant infections. Development of resistance-resistant antibiotics could be achieved via the coordinated networking of clinicians, microbiologists, natural product chemists, and pharmacologists together with pharmaceutical venture capitalist companies.

A systems approach using OSMAC, Log P and NMR fingerprinting: An approach to novelty.

The growing number of sequenced microbial genomes has revealed a remarkably large number of secondary metabolite biosynthetic clusters for which the compounds are still unknown. The aim of the present work was to apply a strategy to detect newly induced natural products by cultivating microorganisms in different fermentation conditions. The metabolomic analysis of 4160 fractions generated from 13 actinomycetes under 32 different culture conditions was carried out by 1H NMR spectroscopy and multivariate analysis. The principal component analysis (PCA) of the 1H NMR spectra showed a clear discrimination between those samples within PC1 and PC2. The fractions with induced metabolites that are only produced under specific growth conditions was identified by PCA analysis. This method allows an efficient differentiation within a large dataset with only one fractionation step. This work demonstrates the potential of NMR spectroscopy in combination with metabolomic data analysis for the screening of large sets of fractions.