Neopetrotaurines A-C, Isoquinoline Alkaloids with an Unprecedented Taurine Bridge from the Sponge Neopetrosia sp.

Neopetrotaurines A-C (1-3), unusual alkaloids possessing two isoquinoline-derived moieties that are linked via a unique taurine bridge, were isolated from a Neopetrosia sp. marine sponge. These new compounds have proton-deficient structural scaffolds that are difficult to unambiguously assign using only conventional 2- and 3-bond 1H-13C and 1H-15N heteronuclear correlation data. Thus, the application of LR-HSQMBC and HMBC NMR experiments optimized to detect 4- and 5-bond long-range 1H-13C heteronuclear correlations facilitated the structure elucidation of these unusual taurine-bridged marine metabolites. Neopetrotaurines A-C (1-3) showed significant inhibition of transcription driven by the oncogenic fusion protein PAX3-FOXO1, which is associated with alveolar rhabdomyosarcoma, and cytotoxic activity against PAX3-FOXO1-positive cell lines.

Rare Caulamidine Hexacyclic Alkaloids from the Marine Ascidian Polyandrocarpa sp.

Two new caulamidines C (2) and D (4) and three isocaulamidines B, C, and D (1, 3, and 5) along with the known compound caulamidine B (6) were isolated from the marine ascidian Polyandrocarpa sp. Their structures were elucidated by analysis of nuclear magnetic resonance (NMR) and electronic circular dichroism (ECD) data. Isocaulamidines have an altered pattern of N-methyl substitution (N-15 vs N-13 in the caulamidines) with a concomitant double-bond rearrangement to provide a new C-14/N-13 imine functionality. Caulamidine C (2) and isocaulamidine C (3) are the first members of this alkaloid family with two chlorine substituents in the core 6H-2,6-naphthyridine ring system.

Identification of natural product modulators of Merkel cell carcinoma cell growth and survival.

Merkel cell carcinoma (MCC) is a rare, but aggressive skin cancer the incidence of which has increased significantly in recent years. The majority of MCCs have incorporated Merkel cell polyomavirus (VP-MCC) while the remainder are virus-negative (VN-MCC). Although a variety of therapeutic options have shown promise in treating MCC, there remains a need for additional therapeutics as well as probes for better understanding MCC. A high-throughput screening campaign was used to assess the ability of > 25,000 synthetic and natural product compounds as well as > 20,000 natural product extracts to affect growth and survival of VN-MCC and VP-MCC cell lines. Sixteen active compounds were identified that have mechanisms of action reported in the literature along with a number of compounds with unknown mechanisms. Screening results with pure compounds suggest a range of potential targets for MCC including DNA damage, inhibition of DNA or protein synthesis, reactive oxygen species, and proteasome inhibition as well as NFκB inhibition while also suggesting the importance of zinc and/or copper binding. Many of the active compounds, particularly some of the natural products, have multiple reported targets suggesting that this strategy might be a particularly fruitful approach. Processing of several active natural product extracts resulted in the identification of additional MCC-active compounds. Based on these results, further investigations focused on natural products sources, particularly of fungal origin, are expected to yield further potentially useful modulators of MCC.

Roseabol A, a New Peptaibol from the Fungus Clonostachys rosea.

A new 11 amino acid linear peptide named roseabol A (1) and the known compound 13-oxo-trans-9,10-epoxy-11(E)-octadecenoic acid (2) were isolated from the fungus Clonostachys rosea. Combined NMR and MS analysis revealed that roseabol A (1) contained amino acid residues characteristic of the peptaibol family of peptides such as isovaline, α-aminoisobutyric acid, hydroxyproline, leucinol, and an N-terminal isovaleric acid moiety. The amino acid sequence was established by a combination of NMR studies and tandem MS fragmentation analyses, and the absolute configurations of the constituent amino acids of 1 were determined by the advanced Marfey's method. Compound 2 showed inhibitory activity against Merkel cell carcinoma, a rare and difficult-to-treat type of skin cancer, with an IC50 value of 16.5 µM.

Sinularamides A-G, Terpenoid-Derived Spermidine and Spermine Conjugates with Casitas B-Lineage Lymphoma Proto-Oncogene B (Cbl-b) Inhibitory Activities from a Sinularia sp. Soft Coral.

An extract of a Sinularia sp. soft coral showed inhibitory activity against the E3-ubiquitin ligase casitas B-lineage lymphoma proto-oncogene B (Cbl-b). Subsequent bioassay-guided separation of the extract provided a series of terpenoid-derived spermidine and spermine amides that were named sinularamides A-G (1-7). Compounds 1-7 represent new natural products; however, sinularamide A (1) was previously reported as a synthetic end product. The structures of sinularamides A-G (1-7) were elucidated by analysis of spectroscopic and spectrometric data from NMR, IR, and HRESIMS experiments and by comparison with literature data. All of the isolated compounds showed Cbl-b inhibitory activities with IC50 values that ranged from approximately 6.5 to 33 µM.

A Molecular Networking Strategy: High-Throughput Screening and Chemical Analysis of Brazilian Cerrado Plant Extracts against Cancer Cells.

Plants have historically been a rich source of successful anticancer drugs and chemotherapeutic agents, with research indicating that this trend will continue. In this contribution, we performed high-throughput cytotoxicity screening of 702 extracts from 95 plant species, representing 40 families of the Brazilian Cerrado biome. Activity was investigated against the following cancer cell lines: colon (Colo205 and Km12), renal (A498 and U031), liver (HEP3B and SKHEP), and osteosarcoma (MG63 and MG63.3). Dose-response tests were conducted with 44 of the most active extracts, with 22 demonstrating IC50 values ranging from <1.3 to 20 µg/mL. A molecular networking strategy was formulated using the Global Natural Product Social Molecular Networking (GNPS) platform to visualize, analyze, and annotate the compounds present in 17 extracts active against NCI-60 cell lines. Significant cytotoxic activity was found for Salacia crassifolia, Salacia elliptica, Simarouba versicolor, Diospyros hispida, Schinus terebinthifolia, Casearia sylvestris var. lingua, Magonia pubescens, and Rapanea guianensis. Molecular networking resulted in the annotation of 27 compounds. This strategy provided an initial overview of a complex and diverse natural product data set, yielded a large amount of chemical information, identified patterns and known compounds, and assisted in defining priorities for further studies.

Sesterterpenoids: chemistry, biology, and biosynthesis.

Covering: July 2012 to December 2019Over the last seven years, expanding research efforts focused on sesterterpenoids has led to the isolation, identification, and characterization of numerous structurally novel and biologically active sesterterpenoids. These newly reported sesterterpenoids provide diverse structures that often incorporate unprecedented ring systems and new carbon skeletons, as well as unusual functional group arrays. Biological activities of potential biomedical importance including suppression of cancer cell growth, inhibition of enzymatic activity, and modulation of receptor signaling, as well as ecologically important functions such as antimicrobial effects and deterrence of herbivorous insects have been associated with a variety of sesterterpenoids. There has also been a rapid growth in our knowledge of the genomics, enzymology, and specific pathways associated with sesterterpene biosynthesis. This has opened up new opportunities for future sesterterpene discovery and diversification through the expression of new cryptic metabolites and the engineered manipulation of associated biosynthetic machinery and processes. In this paper we reviewed 498 new sesterterpenoids, including their structures, source organisms, country of origin, relevant bioactivities, and biosynthesis.

NMR characterization of rearranged staurosporine aglycone analogues from the marine sponge Damiria sp.

The indolocarbazole family of bisindole alkaloids is best known for the natural product staurosporine, a protein kinase C inhibitor that belongs to the indolo[2,3-a]carbazole structural class. A large number of other indolo[2,3-a]carbazoles have subsequently been isolated and identified, but other isomeric forms of indolocarbazole natural products have rarely been reported. An extract of the marine sponge Damiria sp., which represents an understudied genus, provided two novel alkaloids named damirines A (1) and B (2). Their structures were assigned by comprehensive NMR spectroscopic analyses, and for compound 2, this included application of the LR-HSQMBC pulse sequence, a long-range heteronuclear correlation experiment that has particular utility for defining proton-deficient scaffolds. The damirines represent a new hexacyclic carbon-nitrogen framework comprised of an indolo[3,2-a]carbazole fused with either an aminoimidazole or a imidazolone ring. Compound 1 showed selective cytotoxic properties toward six different cell lines in the NCI-60 cancer screen.

Recifin A, Initial Example of the Tyr-Lock Peptide Structural Family, Is a Selective Allosteric Inhibitor of Tyrosyl-DNA Phosphodiesterase I.

Tyrosyl-DNA phosphodiesterase 1 (TDP1) is a molecular target for the sensitization of cancer cells to the FDA-approved topoisomerase inhibitors topotecan and irinotecan. High-throughput screening of natural product extract and fraction libraries for inhibitors of TDP1 activity resulted in the discovery of a new class of knotted cyclic peptides from the marine sponge Axinella sp. Bioassay-guided fractionation of the source extract resulted in the isolation of the active component which was determined to be an unprecedented 42-residue cysteine-rich peptide named recifin A. The native NMR structure revealed a novel fold comprising a four strand antiparallel ß-sheet and two helical turns stabilized by a complex disulfide bond network that creates an embedded ring around one of the strands. The resulting structure, which we have termed the Tyr-lock peptide family, is stabilized by a tyrosine residue locked into three-dimensional space. Recifin A inhibited the cleavage of phosphodiester bonds by TDP1 in a FRET assay with an IC50 of 190 nM. Enzyme kinetics studies revealed that recifin A can specifically modulate the enzymatic activity of full-length TDP1 while not affecting the activity of a truncated catalytic domain of TDP1 lacking the N-terminal regulatory domain (Δ1-147), suggesting an allosteric binding site for recifin A on the regulatory domain of TDP1. Recifin A represents both the first of a unique structural class of knotted disulfide-rich peptides and defines a previously unseen mechanism of TDP1 inhibition that could be productively exploited for potential anticancer applications.

Denigrins and Dactylpyrroles, Arylpyrrole Alkaloids from a Dactylia sp. Marine Sponge.

Seven new arylpyrrole alkaloids (1-7), along with four known compounds, were isolated from an extract of a Dactylia sp. nov. marine sponge, and their structures were elucidated by interpretation of NMR and MS spectroscopic data. Denigrins D-G (1-4) have highly substituted pyrrole or pyrrolone rings in their core structures, while dactylpyrroles A-C (5-7) have tricyclic phenanthrene cores. Due to the proton-deficient nature of these scaffolds, key heteronuclear correlations from 1H-15N HMBC and LR-HSQMBC NMR experiments were used in the structure assignment of denigrin D (1). Dictyodendrin F (8), a previously described co-metabolite, inhibited transcription driven by the oncogenic PAX3-FOXO1 fusion gene with an IC50 value of 13 µM.

Suberitamides A-C, Aryl Alkaloids from a Pseudosuberites sp. Marine Sponge that Inhibit Cbl-b Ubiquitin Ligase Activity.

Three new aryl alkaloids named suberitamides A-C (1-3), were isolated from an extract of the marine sponge Pseudosuberites sp. collected along the coast of North Carolina. Their planar structures were established by extensive nuclear magnetic resonance (NMR) and mass spectrometry (MS) analysis. To assign the challenging relative configuration of the saturated five-membered ring in suberitamide A (1), a simple and efficient NMR protocol was applied that is based on the analysis of 2- and 3-bond 1H-13C spin-spin coupling constants using a PIP (pure in-phase) HSQMBC (heteronuclear single quantum multiple bond correlation) IPAP (in-phase and anti-phase) experiment. Suberitamides A (1) and B (2) inhibited Cbl-b, an E3 ubiquitin ligase that is an important modulator of immune cell function, with IC50 values of approximately 11 µM.

Deciphering the chemical instability of sphaeropsidin A under physiological conditions - degradation studies and structural elucidation of the major metabolite.

The fungal metabolite sphaeropsidin A (SphA) has been recognised for its promising cytotoxicity, particularly towards apoptosis- and multidrug-resistant cancers. Owing to its intriguing activity, the development of SphA as a potential anticancer agent has been pursued. However, this endeavour is compromised since SphA exhibits poor physicochemical stability under physiological conditions. Herein, SphA's instability in biological media was explored utilizing LC-MS. Notably, the degradation tendency was found to be markedly enhanced in the presence of amino acids in the cell medium utilized. Furthermore, the study investigated the presence of degradation adducts, including the identification, isolation and structural elucidation of a major degradation metabolite, (4R)-4,4',4'-trimethyl-3'-oxo-4-vinyl-4',5',6',7'-tetrahydro-3'H-spiro[cyclohexane-1,1'-isobenzofuran]-2-ene-2-carboxylic acid. Considering the reduced cytotoxic potency of aged SphA solutions, as well as that of the isolated degradation metabolite, the reported antiproliferative activity has been attributed primarily to the parent compound (SphA) and not its degradation species. The fact that SphA continues to exhibit remarkable bioactivity, despite being susceptible to degradation, motivates future research efforts to address the challenges associated with this instability impediment.

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.

Swinhopeptolides A and B: Cyclic Depsipeptides from the Sponge Theonella swinhoei That Inhibit Ras/Raf Interaction.

Two new cyclic depsipeptides named swinhopeptolides A (1) and B (2) have been isolated from the marine sponge Theonella swinhoei cf. verrucosa, collected from Papua New Guinea. They each contain 11 diverse amino acid residues and 13-carbon polyketide moieties attached at the N-terminus. Compounds 1 and 2 each exist as two conformers in DMSO-d6 due to cis/trans isomerism of the proline residue, and their structures were successfully assigned by extensive NMR analyses complemented by chemical degradation and derivatization studies. Swinhopeptolide B (2) contains a previously undescribed 2,6,8-trimethyldeca-(2E,4E,6E)-trienoic acid moiety N-linked to a terminal serine residue. Swinhopeptolides A (1) and B (2) showed significant inhibition of the Ras/Raf signaling pathway with IC50 values of 5.8 and 8.5 µM, respectively.

Structure elucidation and absolute configuration of metabolites from the soil-derived fungus Dictyosporium digitatum using spectroscopic and computational methods.

Following the discovery of a new class of compounds that inhibit the mucosa-associated lymphoid tissue lymphoma translocation 1 (MALT1) protease in a prior study, further chemical investigation of the Dictyosporium digitatum fungus resulted in the identification of 16 additional metabolites, including 12 undescribed compounds (1-12). The constitution and relative configuration of these new molecules were established by comprehensive NMR and HRMS analyses. Their absolute configurations were determined by employing Mosher's ester analysis and TDDFT ECD calculations. Two sesquiterpenes, dictyosporins A (1) and B (2), possess an undescribed eudesmen-type of structural scaffold. The ability of the isolated compounds to inhibit MALT1 proteolytic activity was evaluated, but none of them exhibited significant inhibition.

Botryllamide G is an ABCG2 inhibitor that improves lapatinib delivery in mouse brain.

Introduction: Transporters comprising the blood-brain barrier complicate delivery of many therapeutics to the central nervous system. The present study ascertained whether the natural product botryllamide G is viable for in vivo inhibition of ABCG2 using lapatinib as a probe for ABCB1 and ABCG2-mediated efflux from the brain. Methods: Wild-type and Mdr1a/Mdr1b (-/-) mice were treated with botryllamide G and lapatinib ("doublet therapy"), and while a separate cohort of wild-type mice was treated with botryllamide, tariquidar and lapatinib ("triplet therapy"). Results: Botryllamide G demonstrates biphasic elimination with a rapid distribution, decreasing below the in vitro IC50 of 6.9 µM within minutes, yet with a relatively slower terminal half-life (4.6 h). In Mdr1a/Mdr1b (-/-) mice, doublet therapy resulted in a significant increase in brain lapatinib AUC at 8 h (2058 h*ng/mL vs 4007 h*ng/mL; P = .031), but not plasma exposure (P = .15). No significant differences were observed after 24 h. Lapatinib brain exposure was greater through 1 h when wild-type mice were administered triplet therapy (298 h*pg/mg vs 120 h*pg/mg; P < .001), but the triplet decreased brain AUC through 24 h vs. mice administered lapatinib alone (2878 h*pg/mg vs 4461hr*ng/mL; P < .001) and did not alter the brain:plasma ratio. Conclusions: In summary, the ABCG2 inhibitor, botryllamide G, increases brain exposure to lapatinib in mice lacking Abcb1, although the combination of botryllamide G and tariquidar increases brain exposure in wild-type mice only briefly (1 h). Additional research is needed to find analogs of this compound that have better pharmacokinetics and pharmacodynamic effects on ABCG2 inhibition.

Characterization by Empirical and Computational Methods of Dictyospiromide, an Intriguing Antioxidant Alkaloid from the Marine Alga Dictyota coriacea.

The challenging structural motif of dictyospiromide (1), a spirosuccinimide alkaloid with antioxidant properties that are associated with activation of the Nrf2/ARE signaling pathway, was assigned using contemporary NMR experiments complemented with anisotropic NMR, chiroptical, and computational methodologies. Anisotropic NMR parameters provided critical orthogonal verification of the configuration of the difficult to assign spiro carbon and the other stereogenic centers in 1.

Madecassic Acid Derivatives as Potential Anticancer Agents: Synthesis and Cytotoxic Evaluation.

A series of novel madecassic acid (1) derivatives was synthesized, and their cytotoxicity was evaluated against the NCI-60 panel of cancer cell lines. Several analogues exhibited broad-spectrum cytotoxic activities over all nine tumor types represented in the panel, with more potent antiproliferative activities observed against selected cancer cell lines, including multidrug-resistant phenotypes. Among them, compound 29 showed GI50 (50% growth inhibition) values ranging from 0.3 to 0.9 µM against 26 different tumor cell lines and selectivity for one colon (COLO 205) and two melanoma (SK-MEL-5 and UACC-257) cell lines at the TGI (total growth inhibition) level. The mode of action of 29 was predicted by CellMiner bioinformatic analysis and confirmed by biochemical and cell-based experiments to involve inhibition of the DNA replication process, particularly the initiation of replication, and disruption of mitochondrial membrane potential. The present findings suggest this novel madecassic acid derivative may have potential as an anticancer therapeutic lead for both solid and hematological tumors.

Secondary Metabolites from the Fungus Dictyosporium sp. and Their MALT1 Inhibitory Activities.

Bioassay-guided separation of an extract from a Dictyosporium sp. isolate led to the identification of six new compounds, 1-6, together with five known compounds, 7-11. The structures of the new compounds were primarily established by extensive 1D and 2D NMR experiments. The absolute configurations of compounds 3-6 were determined by comparison of their experimental electronic circular dichroism (ECD) spectra with DFT quantum mechanical calculated ECD spectra. Compounds 3-5 possess novel structural scaffolds, and biochemical studies revealed that oxepinochromenones 1 and 7 inhibited the activity of MALT1 protease.

Harnessing Natural Product Diversity for Fluorophore Discovery: Naturally Occurring Fluorescent Hydroxyanthraquinones from the Marine Crinoid Pterometra venusta.

Fluorescent small molecules are important tools in many aspects of modern biology. A two-stage evaluation process involving fluorescence screening and live-cell imaging was developed to facilitate the identification of new fluorescent probes from extracts housed within the NCI Natural Products Repository. To this end, over 2000 extracts and prefractionated samples were examined, including an extract from the marine crinoid Pterometra venusta. An optically guided evaluation involving stepwise fluorescence screening and live-cell imaging was developed to enable the isolation of fluorescent natural products. These efforts resulted in the isolation of six hydroxyanthraquinone compounds, three of which are new natural products. These purified metabolites were examined for their potential as cellular imaging probes, and they demonstrate that natural product libraries can be a good source of new fluorescent agents.