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.

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.

Elucidation of Spirodactylone, a Polycyclic Alkaloid from the Sponge Dactylia sp., and Nonenzymatic Generation from the Co-metabolite Denigrin B.

Spirodactylone (1), a hexacyclic indolizidone alkaloid possessing a novel spiro ring system, was isolated from the marine sponge Dactylia sp. The structure was elucidated by extensive spectroscopic methods including application of the LR-HSQMBC NMR pulse sequence. Oxidative cyclization of denigrin B (2), an aryl-substituted 2-oxo-pyrroline derivative that was also isolated from the sponge extract, provided material identical to spirodactylone (1). This confirmed the assigned structure and provides insight into the probable biogenesis of 1.

Unequivocal determination of caulamidines A and B: application and validation of new tools in the structure elucidation tool box.

Ambiguities and errors in the structural assignment of organic molecules hinder both drug discovery and total synthesis efforts. Newly described NMR experimental approaches can provide valuable structural details and a complementary means of structure verification. The caulamidines are trihalogenated alkaloids from a marine bryozoan with an unprecedented structural scaffold. Their unique carbon and nitrogen framework was deduced by conventional NMR methods supplemented by new experiments that define 2-bond heteronuclear connectivities, reveal very long-range connectivity data, or visualize the 35,37Cl isotopic effect on chlorinated carbons. Computer-assisted structural elucidation (CASE) analysis of the spectroscopic data for caulamidine A provided only one viable structural alternative. Anisotropic NMR parameters, specifically residual dipolar coupling and residual chemical shift anisotropy data, were measured for caulamidine A and compared to DFT-calculated values for the proposed structure, the CASE-derived alternative structure, and two energetically feasible stereoisomers. Anisotropy-based NMR experiments provide a global, orthogonal means to verify complex structures free from investigator bias. The anisotropic NMR data were fully consistent with the assigned structure and configuration of caulamidine A. Caulamidine B has the same heterocyclic scaffold as A but a different composition and pattern of halogen substitution. Caulamidines A and B inhibited both wild-type and drug-resistant strains of the malaria parasite Plasmodium falciparum at low micromolar concentrations, yet were nontoxic to human cells.

Deguelins, Natural Product Modulators of NF1-Defective Astrocytoma Cell Growth Identified by High-Throughput Screening of Partially Purified Natural Product Extracts.

A high-throughput screening assay for modulators of Trp53/NF1 mutant astrocytoma cell growth was adapted for use with natural product extracts and applied to a novel collection of prefractionated/partially purified extracts. Screening 68 427 samples identified active fractions from 95 unique extracts, including the terrestrial plant Millettia ichthyotona. Only three of these extracts showed activity in the crude extract form, thus demonstrating the utility of a partial purification approach for natural product screening. The NF1 screening assay was used to guide purification of active compounds from the M. ichthyotona extract, which yielded the two rotenones deguelin (1) and dehydrodeguelin (2). The deguelins have been reported to affect growth of a number of cancer cell lines. They potently inhibited growth of only one of a panel of NF1/Trp53 mutant murine astrocytoma cell lines, possibly related to epigenetic factors, but had no effect on the growth of normal astrocytes. These results suggest the potential utility of deguelins as tools for further investigating NF1 astrocytoma cell growth. These bioprobes were identified only as a result of screening partially purified natural product extracts.

Stellettapeptins A and B, HIV-inhibitory cyclic depsipeptides from the marine sponge Stelletta sp.

Two new HIV-inhibitory depsipeptides, stellettapeptins A (1) and B (2), were isolated from an extract of the marine sponge Stelletta sp., collected from northwestern Australia. Structures of these cyclic nonribosomal peptides were elucidated on the basis of extensive NMR data analysis, and chemical degradation and derivatization studies. Stellettapeptins contain numerous nonproteinogenic amino acid residues and they are the first peptides reported to contain a 3-hydroxy-6,8-dimethylnon-4-(Z)-enoic acid moiety. Compounds 1 and 2 potently inhibit infection of human T-lymphoblastoid cells by HIV-1RF with EC50 values of 23 and 27 nM, respectively.

Identification of compounds by high-content screening that induce cytoplasmic to nuclear localization of a fluorescent estrogen receptor α chimera and exhibit agonist or antagonist activity in vitro.

We have completed a robust high-content imaging screen for novel estrogen receptor α (ERα) agonists and antagonists by quantitation of cytoplasmic to nuclear translocation of an estrogen receptor chimera in 384-well plates. The screen was very robust, with Z' values >0.7 and coefficients of variation (CV) <5%. The screen utilized a stably transfected green fluorescent protein-tagged glucocorticoid/estrogen receptor (GFP-GRER) chimera, which consisted of the N-terminus of the glucocorticoid receptor fused to the human ERα ligand binding domain. The GFP-GRER exhibited cytoplasmic localization in the absence of ERα ligands and translocated to the nucleus in response to stimulation with ERα agonists and antagonists. The BD Pathway 435 imaging system was used for image acquisition, analysis of translocation dynamics, and cytotoxicity measurements. We screened 224,891 samples from our synthetic, pure natural product libraries, prefractionated natural product extracts library, and crude natural product extracts library, which produced a 0.003% hit rate. In addition to identifying several known ER ligands, five compounds were discovered that elicited significant activity in the screen. Transactivation potential studies demonstrated that two hit compounds behave as agonists, while three compounds elicited antagonist activity in MCF-7 cells.

Inhibition of ABCG2-mediated drug efflux by naphthopyrones from marine crinoids.

Five new naphthopyrones (1-5) along with the known compounds TMC-256A1, 5,8-dihydroxy-6-methoxy-2-propyl-4H-naphtho[2,3-b]pyran-4-one, TMC-256C1, comaparvin, 6-methoxycomaparvin, and 6-methoxycomaparvin 5-methyl ether (6-11) were isolated from crinoids of the family Comasteridae. All compounds were tested for their ability to inhibit the multidrug transporter ABCG2, which plays a role in drug resistance. Six of the seven angular naphthopyrones showed moderate activity with <60% inhibition of ABCG2-mediated transport as compared to the positive control fumitremorgin C. None of the linear naphthopyrones inhibited ABCG2-mediated efflux.

Neamphamide A, a new HIV-inhibitory depsipeptide from the Papua New Guinea marine sponge Neamphius huxleyi.

A new HIV-inhibitory cyclic depsipeptide, neamphamide A (2), was isolated from a Papua New Guinea collection of the marine sponge Neamphius huxleyi. Its structure was established through interpretation of spectroscopic data and by acid hydrolysis, derivatization of the free amino acids, and LC-MS analysis of the derivatives. Neamphamide A (2) contains 11 amino acid residues and an amide-linked 3-hydroxy-2,4,6-trimethylheptanoic acid moiety. The amino acid constituents were identified as L-Leu, L-NMeGln, D-Arg, D- and L-Asn, two residues of D-allo-Thr, L-homoproline, (3S,4R)-3,4-dimethyl-L-glutamine, beta-methoxytyrosine, and 4-amino-7-guanidino-2,3-dihydroxyheptanoic acid. In a cell-based XTT assay, 2 exhibited potent cytoprotective activity against HIV-1 infection with an EC50 of approximately 28 nM.

Structures of the complexes of a potent anti-HIV protein cyanovirin-N and high mannose oligosaccharides.

The development of anti-human immunodeficiency virus (HIV) microbicides for either topical or ex vivo use is of considerable interest, mainly due to the difficulties in creating a vaccine that would be active against multiple clades of HIV. Cyanovirin-N (CV-N), an 11-kDa protein from the cyanobacterium (blue-green algae) Nostoc ellipsosporum with potent virucidal activity, was identified in the search for such antiviral agents. The binding of CV-N to the heavily glycosylated HIV envelope protein gp120 is carbohydrate-dependent. Since previous CV-N-dimannose structures could not fully explain CV-N-oligomannose binding, we determined the crystal structures of recombinant CV-N complexed to Man-9 and a synthetic hexamannoside, at 2.5- and 2.4-A resolution, respectively. CV-N is a three-dimensional domain-swapped dimer in the crystal structures with two primary sites near the hinge region and two secondary sites on the opposite ends of the dimer. The binding interface is constituted of three stacked alpha1-->2-linked mannose rings for Man-9 and two stacked mannose rings for hexamannoside with the rest of the saccharide molecules pointing to the solution. These structures show unequivocally the binding geometry of high mannose sugars to CV-N, permitting a better understanding of carbohydrate binding to this potential new lead for the design of drugs against AIDS.

Domain-swapped structure of a mutant of cyanovirin-N.

Cyanovirin-N (CV-N) is a potent 11 kDa HIV-inactivating protein that binds with high affinity to the HIV surface envelope protein gp120. A double mutant P51S/S52P of CV-N was engineered by swapping two critical hinge-region residues Pro51 and Ser52. This mutant has biochemical and biophysical characteristics equivalent to the wild-type CV-N and its structure resembles that of wild-type CV-N. However, the mutant shows a different orientation in the hinge region that connects two domains of the protein. The observation that this double mutant crystallizes under a wide variety of conditions challenges some of the current hypotheses on domain swapping and on the role of hinge-region proline residues in domain orientation. The current structure contributes to the understanding of domain swapping in cyanovirins, permitting rational design of domain-swapped CV-N mutants.