Isolation, Structural Analysis and Biological Activity Assays of Biselisabethoxanes A and B: Two Dissymmetric Bis-Diterpenes from the Southwestern Caribbean Sea Gorgonian Coral Pseudopterogorgia elisabethae.

Two novel dissymmetric diterpenoids, biselisabethoxanes A and B (1 and 2), were isolated from the hexane extracts of the gorgonian coral Pseudopterogorgia elisabethae. Biselisabethoxane A (1) represents the first example of a marine-derived C40 dimer made of two distinct diterpene fragments, whereas biselisabethoxane B (2) is a fused heterodimer stemming from coupling of two amphilectane-based fragments. The structures of 1 and 2 were elucidated based on 1D and 2D NMR spectral data analysis. The molecular structure of 1 was subsequently confirmed by X-ray crystallographic analysis. When evaluated for their inhibitory effects in a series of well-established biological activity assays the isolated compounds were shown to moderately inhibit the growth of Mycobacterium tuberculosis.

Marine pharmacology in 2018: Marine compounds with antibacterial, antidiabetic, antifungal, anti-inflammatory, antiprotozoal, antituberculosis and antiviral activities; affecting the immune and nervous systems, and other miscellaneous mechanisms of action.

The 2018 marine pharmacology literature review represents a continuation of the previous 11 reviews of a series initiated in 1998. Preclinical marine pharmacology research during 2018 was performed by investigators in 44 countries and contributed novel pharmacology for 195 marine compounds. The peer-reviewed marine natural products pharmacology literature reported antibacterial, antifungal, antiprotozoal, antituberculosis, and antiviral activities for 53 compounds, 73 compounds which presented antidiabetic and anti-inflammatory activities as well as affecting the immune and nervous system, while in contrast 69 compounds were reported to show miscellaneous mechanisms of action which may contribute upon further investigation to several pharmacological classes. Thus, in 2018, the preclinical marine natural product pharmacology pipeline continued to report novel pharmacology as well as new lead compounds for the clinical marine pharmaceutical pipeline, which currently contributes to therapeutic strategies for several disease categories.

Plakortinic acids C and D: a pair of peroxide-polyketides possessing a rare 7,8-dioxatricyclo[4.2.2.02,5]dec-9-ene core from a two-sponge association of Plakortis symbiotica-Xestospongia deweerdtae.

Plakortinic acids C (3) and D (4), two unprecedented peroxide-polyketides with 7,8-dioxatricyclo[4.2.2.02,5]dec-9-ene scaffold, as well as known biogenetically related congeners, plakortinic acids A (1) and B (2), were isolated from a two-sponge association of Plakortis symbiotica-Xestospongia deweerdtae. Upon chemical derivatization, the structures and relative configurations of 3 and 4 were characterized by analysis of HRESIMS and NMR spectroscopic data, molecular modeling studies, and chiroptical comparisons with known natural products and published values of [α]D of related synthetic analogs. A mixture of methyl ester derivatives 5 and 6 displayed negligible cytotoxicity against a panel of 60 cell lines of various human cancers at a concentration of 10 µM.

Marine Pharmacology in 2016-2017: Marine Compounds with Antibacterial, Antidiabetic, Antifungal, Anti-Inflammatory, Antiprotozoal, Antituberculosis and Antiviral Activities; Affecting the Immune and Nervous Systems, and Other Miscellaneous Mechanisms of Action.

The review of the 2016-2017 marine pharmacology literature was prepared in a manner similar as the 10 prior reviews of this series. Preclinical marine pharmacology research during 2016-2017 assessed 313 marine compounds with novel pharmacology reported by a growing number of investigators from 54 countries. The peer-reviewed literature reported antibacterial, antifungal, antiprotozoal, antituberculosis, and antiviral activities for 123 marine natural products, 111 marine compounds with antidiabetic and anti-inflammatory activities as well as affecting the immune and nervous system, while in contrast 79 marine compounds displayed miscellaneous mechanisms of action which upon further investigation may contribute to several pharmacological classes. Therefore, in 2016-2017, the preclinical marine natural product pharmacology pipeline generated both novel pharmacology as well as potentially new lead compounds for the growing clinical marine pharmaceutical pipeline, and thus sustained with its contributions the global research for novel and effective therapeutic strategies for multiple disease categories.

The Discreet Structural Diversity of Briarellins: DU8+ Guided Multiple Structure Revisions Yielded Two Unknown Structural Types.

Briarellins, a subset of C2-C11 cyclized cembranoids, were proposed to contain a C3-C14 ether or lactone bridge, similar to asbestinins. However, the total synthesis of the proposed structure of briarellin J revealed a misassignment. We revisited briarellins, computationally, with the help of a recently developed hybrid DFT/parametric method, DU8+, and revised the structures of briarellin C14-C3 ε-lactones to new structural types containing either a C14-C11 or C14-C12 lactone bridge. The original structures of briarellin and asbestinin ethers were confirmed.

A comparative study of small molecules targeting eIF4A.

The PI3K/Akt/mTOR kinase pathway is extensively deregulated in human cancers. One critical node under regulation of this signaling axis is eukaryotic initiation factor (eIF) 4F, a complex involved in the control of translation initiation rates. eIF4F-dependent addictions arise during tumor initiation and maintenance due to increased eIF4F activity-generally in response to elevated PI3K/Akt/mTOR signaling flux. There is thus much interest in exploring eIF4F as a small molecule target for the development of new anticancer drugs. The DEAD-box RNA helicase, eIF4A, is an essential subunit of eIF4F, and several potent small molecules (rocaglates, hippuristanol, pateamine A) affecting its activity have been identified and shown to demonstrate anticancer activity in vitro and in vivo in preclinical models. Recently, a number of new small molecules have been reported as having the capacity to target and inhibit eIF4A. Here, we undertook a comparative analysis of their biological activity and specificity relative to the eIF4A inhibitor, hippuristanol.

Searching for Small Molecules with an Atomic Sort.

The discovery of biologically active small molecules requires sifting through large amounts of data to identify unique or unusual arrangements of atoms. Here, we develop, test and evaluate an atom-based sort to identify novel features of secondary metabolites and demonstrate its use to evaluate novelty in marine microbial and sponge extracts. This study outlines an important ongoing advance towards the translation of autonomous systems to identify, and ultimately elucidate, atomic novelty within a complex mixture of small molecules.

Marine Pharmacology in 2014-2015: Marine Compounds with Antibacterial, Antidiabetic, Antifungal, Anti-Inflammatory, Antiprotozoal, Antituberculosis, Antiviral, and Anthelmintic Activities; Affecting the Immune and Nervous Systems, and Other Miscellaneous Mechanisms of Action.

The systematic review of the marine pharmacology literature from 2014 to 2015 was completed in a manner consistent with the 1998-2013 reviews of this series. Research in marine pharmacology during 2014-2015, which was reported by investigators in 43 countries, described novel findings on the preclinical pharmacology of 301 marine compounds. These observations included antibacterial, antifungal, antiprotozoal, antituberculosis, antiviral, and anthelmintic pharmacological activities for 133 marine natural products, 85 marine compounds with antidiabetic, and anti-inflammatory activities, as well as those that affected the immune and nervous system, and 83 marine compounds that displayed miscellaneous mechanisms of action, and may probably contribute to novel pharmacological classes upon further research. Thus, in 2014-2015, the preclinical marine natural product pharmacology pipeline provided novel pharmacology as well as new lead compounds for the clinical marine pharmaceutical pipeline, and thus continued to contribute to ongoing global research for alternative therapeutic approaches to many disease categories.

Tracing MYC Expression for Small Molecule Discovery.

Our inability to effectively "drug" targets such as MYC for therapeutic purposes requires the development of new approaches. We report on the implementation of a phenotype-based assay for monitoring MYC expression in multiple myeloma cells. The open reading frame (ORF) encoding an unstable variant of GFP was engineered immediately downstream of the MYC ORF using CRISPR/Cas9, resulting in co-expression of both proteins from the endogenous MYC locus. Using fluorescence readout as a surrogate for MYC expression, we implemented a pilot screen in which ∼10,000 compounds were prosecuted. Among known MYC expression inhibitors, we identified cardiac glycosides and cytoskeletal disruptors to be quite potent. We demonstrate the power of CRISPR/Cas9 engineering in establishing phenotype-based assays to identify gene expression modulators.

Target-Based Screening against eIF4A1 Reveals the Marine Natural Product Elatol as a Novel Inhibitor of Translation Initiation with In Vivo Antitumor Activity.

Purpose: The DEAD-box RNA helicase eIF4A1 carries out the key enzymatic step of cap-dependent translation initiation and is a well-established target for cancer therapy, but no drug against it has entered evaluation in patients. We identified and characterized a natural compound with broad antitumor activities that emerged from the first target-based screen to identify novel eIF4A1 inhibitors.Experimental Design: We tested potency and specificity of the marine compound elatol versus eIF4A1 ATPase activity. We also assessed eIF4A1 helicase inhibition, binding between the compound and the target including binding site mutagenesis, and extensive mechanistic studies in cells. Finally, we determined maximum tolerated dosing in vivo and assessed activity against xenografted tumors.Results: We found elatol is a specific inhibitor of ATP hydrolysis by eIF4A1 in vitro with broad activity against multiple tumor types. The compound inhibits eIF4A1 helicase activity and binds the target with unexpected 2:1 stoichiometry at key sites in its helicase core. Sensitive tumor cells suffer acute loss of translationally regulated proteins, leading to growth arrest and apoptosis. In contrast to other eIF4A1 inhibitors, elatol induces markers of an integrated stress response, likely an off-target effect, but these effects do not mediate its cytotoxic activities. Elatol is less potent in vitro than the well-studied eIF4A1 inhibitor silvestrol but is tolerated in vivo at approximately 100× relative dosing, leading to significant activity against lymphoma xenografts.Conclusions: Elatol's identification as an eIF4A1 inhibitor with in vivo antitumor activities provides proof of principle for target-based screening against this highly promising target for cancer therapy. Clin Cancer Res; 24(17); 4256-70. ©2018 AACR.

Synthesis and biological analysis of truncated calyculone H.

Herein, we report for the first time the design and linear synthesis of a truncated calyculone H (7) that lacks the telltale isopropyl/isopropylene groups, whereas the 12-membered macrocycle remains intact. Key steps for the framework of target molecule include allylic oxidation using SeO2, Sharpless asymmetric epoxidation, Barbier zinc allylation, and ring-closing metathesis (RCM) reactions. A second truncated "calyculone-like" analogue, 27, with a different oxidation pattern around the ring was also synthesized following a similar strategy. Screening for in vitro cytotoxicity against a panel of 60 human cancer cell lines revealed that 7 was as potent if not more so (for a few cell lines) than the natural product calyculone A (2).

Marine Pharmacology in 2012-2013: Marine Compounds with Antibacterial, Antidiabetic, Antifungal, Anti-Inflammatory, Antiprotozoal, Antituberculosis, and Antiviral Activities; Affecting the Immune and Nervous Systems, and Other Miscellaneous Mechanisms of Action.

The peer-reviewed marine pharmacology literature from 2012 to 2013 was systematically reviewed, consistent with the 1998-2011 reviews of this series. Marine pharmacology research from 2012 to 2013, conducted by scientists from 42 countries in addition to the United States, reported findings on the preclinical pharmacology of 257 marine compounds. The preclinical pharmacology of compounds isolated from marine organisms revealed antibacterial, antifungal, antiprotozoal, antituberculosis, antiviral and anthelmitic pharmacological activities for 113 marine natural products. In addition, 75 marine compounds were reported to have antidiabetic and anti-inflammatory activities and affect the immune and nervous system. Finally, 69 marine compounds were shown to display miscellaneous mechanisms of action which could contribute to novel pharmacological classes. Thus, in 2012-2013, the preclinical marine natural product pharmacology pipeline provided novel pharmacology and lead compounds to the clinical marine pharmaceutical pipeline, and contributed significantly to potentially novel therapeutic approaches to several global disease categories.

ATP-competitive, marine derived natural products that target the DEAD box helicase, eIF4A.

Activation of translation initiation is a common trait of cancer cells. Formation of the heterotrimeric eukaryotic initiation factor F (eIF4F) complex is the rate-limiting step in 5' m7GpppN cap-dependent translation. This trimeric complex includes the eIF4E cap binding protein, the eIF4G scaffolding protein, and the DEAD box RNA helicase eIF4A. eIF4A is an ATP-dependent helicase and because it is the only enzyme in the eIF4F complex, it has been shown to be a potential therapeutic target for a variety of malignancies. To this end, we have used a simple ATPase biochemical screen to survey several hundred marine and terrestrial derived natural products. Herein, we report the discovery of two natural products from marine sources, elisabatin A (1) and allolaurinterol (2), which show low µM inhibition of eIF4A ATPase activity. Enzymological analyses revealed 1 and 2 to be ATP-competitive, and cellular evaluations showed reasonable cytotoxicity against A549 (lung cancer) and MDA-MA-468 (breast cancer) cell lines. However, only compound 2 showed potent inhibition of helicase activity congruent with its ATPase inhibitory activity.

Exploring the Sponge Consortium Plakortis symbiotica-Xestospongia deweerdtae as a Potential Source of Antimicrobial Compounds and Probing the Pharmacophore for Antituberculosis Activity of Smenothiazole A by Diverted Total Synthesis.

Fractionation of the bioactive CHCl3-MeOH (1:1) extracts obtained from two collections of the sponge consortium Plakortis symbiotica-Xestospongia deweerdtae from Puerto Rico provided two new plakinidone analogues, designated as plakinidone B (2) and plakinidone C (3), as well as the known plakinidone (1), plakortolide F (4), and smenothiazole A (5). The structures of 1-5 were characterized on the basis of 1D and 2D NMR spectroscopic, IR, UV, and HRMS analysis. The absolute configurations of plakinidones 2 and 3 were established through chemical correlation methods, VCD/ECD experiments, and spectroscopic data comparisons. When assayed in vitro against Mycobacterium tuberculosis H37Rv, none of the plakinidones 1-3 displayed significant activity, whereas smenothiazole A (5) was the most active compound, exhibiting an MIC value of 4.1 µg/mL. Synthesis and subsequent biological screening of 8, a dechlorinated version of smenothiazole A, revealed that the chlorine atom in 5 is indispensable for anti-TB activity.

Plakortinic Acids A and B: Cytotoxic Cycloperoxides with a Bicyclo[4.2.0]octene Unit from Sponges of the Genera Plakortis and Xestospongia.

Plakortinic acids A (2) and B (3), two polyketide endoperoxides with a bicyclo[4.2.0]octene unit, were isolated as minor constituents from the sponge-sponge symbiotic association Plakortis halichondrioides-Xestospongia deweerdtae, along with known epiplakinic acid F (1). The structures of the mixture of two inseparable compounds were determined by spectroscopic analysis. Screening for cytotoxic activity of the mixture against two human tumor cell lines revealed that these compounds are very active at sub-micromolar concentration.

Natural product-based synthesis of novel anti-infective isothiocyanate- and isoselenocyanate-functionalized amphilectane diterpenes.

The marine natural product (-)-8,15-diisocyano-11(20)-amphilectene (1), isolated from the Caribbean sponge Svenzea flava, was used as scaffold to synthetize five new products, all of which were tested against laboratory strains of Plasmodium falciparum and Mycobacterium tuberculosis H37Rv. The scaffold contains two isocyanide units that are amenable to chemical manipulation, enabling them to be elaborated into a small library of sulfur and selenium compounds. Although most of the analogs prepared were less potent than the parent compound, 5 was nearly equipotent showing IC50 values of 0.0066 µM and 0.0025 µM, respectively, against two strains (Dd2 and 3D7) of the malaria parasite. On the other hand, when assayed against the tuberculosis bacterium, analogs 5 and 6 were found to be more potent than 1.

Novel Very Long-Chain α-Methoxylated Δ5,9 Fatty Acids from the Sponge Asteropus niger Are Effective Inhibitors of Topoisomerases IB.

The novel fatty acids (2R,5Z,9Z)-2-methoxy-25-methyl-5,9-hexacosadienoic acid (1a) and (2R,5Z,9Z)-2-methoxy-24-methyl-5,9-hexacosadienoic acid (1b) were isolated in 80 % purity from the Caribbean sponge Asteropus niger by chloroform/methanol extraction followed by solvent partitioning and silica gel column chromatography. The compounds were characterized by utilizing a combination of gas chromatography-mass spectrometry, nuclear magnetic resonance, and circular dichroism. Acids 1a and 1b were not detected in the phospholipids (PtdCho and PtdIns) of the sponge, but rather as free FA and possibly in glycosylceramides. The mixtures of 1a and 1b displayed cytotoxicity towards THP-1 and HepG2 cells with EC50's between 41 and 35 µg/mL. Apoptosis was not the preferred mode of cell death induced by 1a-1b in the THP-1 cells. This implies other types of cytotoxicity mechanisms, such as membrane disruption and/or the inhibition (EC50 = 1.8 µg/mL) of the human topoisomerase IB enzyme (hTopIB), with a mechanism of inhibition different from the one displayed by camptothecin (CPT). In a separate experiment, the mixture of 1a and 1b also displayed cytotoxicity towards ex vivo mouse splenocytes infected with Leishmania infantum amastigotes (IC(50) = 0.17 mg/mL) and free living promastigotes (IC(50) = 0.34 mg/mL). It was also found that the FA were inhibitory of the Leishmania topoisomerase IB (LTopIB) with an EC(50) = 5.1 µg/mL. Taken together, 1a and 1b represent a new class of FA with potential as TopIB inhibitors that preferentially inhibit hTopIB over LTopIB.

Reassignment of the absolute configuration of plakinidone from the sponge consortium Plakortis halichondrioides-Xestospongia deweerdtae using a combination of synthesis and a chiroptical approach.

Recent work by Wu et al. in connection with the first synthesis of the marine natural product plakinidone revealed that the most salient feature of its purported structure, a six-membered perlactone moiety, was in fact a five-membered lactone, i.e. a 3-methyl-4-hydroxy-2(5H)-furanone or tetronic acid ring. With the planar structure of plakinidone confidently revised, we undertook a new investigation to unambiguously establish its absolute configuration. Upon preparing two stable derivatives 1 and 5 from a sample of naturally occurring plakinidone extracted from the sponge association Plakortis halichondriodes-Xetospongia deweerdtae, the absolute configuration was assigned by synthesis and vibrational and electronic circular dichroism (VCD and ECD) measurements in combination with density functional theory calculations at the B3LYP/aug-cc-pVDZ/PCM(CH3CN) level of theory. Our combined efforts and the agreement between the experimental and calculated VCD/ECD spectra of 1 revealed that the absolute configuration of plakinidone was in fact (11S,17R) and not the formerly reported (11S,17S) diastereomer assigned by Wu et al.. Therefore, we propose that natural plakinidone is accurately represented by structure 12.

Structures, semisyntheses, and absolute configurations of the antiplasmodial α-substituted ß-lactam monamphilectines B and C from the sponge Svenzea flava.

Bioassay-guided fractionation of the Caribbean sponge Svenzea flava collected near Mona Island, off the west coast of Puerto Rico, led to the isolation of two isocyanide amphilectane-type diterpenes named monamphilectines B and C (2 and 3). Attached to the backbone of each of these compounds is the first α-substituted monocyclic ß-lactam ring to be isolated from a marine organism. The molecular structures of 2 and 3 were established by spectroscopic methods and then confirmed unequivocally by chemical correlation and comparison of physical and chemical data with the natural products. The new ß-lactams were successfully synthesized in one step, starting from the known diisocyanide 4, via parallel Ugi four-center three-component reactions (U-4C-3CR) that also established their absolute stereostructures. Interestingly, compounds 2 and 3 exhibited activities in the low nanomolar range against the human malaria parasite Plasmodium falciparum.