Portobelamides A and B and Caciqueamide, Cytotoxic Peptidic Natural Products from a Caldora sp. Marine Cyanobacterium.

Three new compounds, portobelamides A and B (1 and 2), 3-amino-2-methyl-7-octynoic acid (AMOYA) and hydroxyisovaleric acid (Hiva) containing cyclic depsipeptides, and one long chain lipopeptide caciqueamide (3), were isolated from a field-collection of a Caldora sp. marine cyanobacterium obtained from Panama as part of the Panama International Cooperative Biodiversity Group Program. Their planar structures were elucidated through analysis of 2D NMR and MS data, especially high resolution (HR) MS2/MS3 fragmentation methods. The absolute configurations of compounds 1 and 2 were deduced by traditional hydrolysis, derivative formation, and chromatographic analyses compared with standards. Portobelamide A (1) showed good cytotoxicity against H-460 human lung cancer cells (33% survival at 0.9 µM).

Synthesis, Cytotoxicity, and Genotoxicity of 10-Aza-9-oxakalkitoxin, an N,N,O-Trisubstituted Hydroxylamine Analog, or Hydroxalog, of a Marine Natural Product.

We describe the synthesis of 10-aza-9-oxakalkitoxin, an N,N,O-trisubstituted hydroxylamine-based analog, or hydroxalog, of the cytotoxic marine natural product kalkitoxin in which the -NMe-O- moiety replaces a -CHMe-CH2- unit in the backbone of the natural product. 10-Aza-9-oxakalkitoxin displays potent and selective cytotoxicity (IC50 2.4 ng mL-1) comparable to that of kalkitoxin itself (IC50 3.2 ng mL-1) against the human hepato-carcinoma cell line HepG2 over both the human leukemia cell line CEM and the normal hematopoietic CFU-GM. Like kalkitoxin, and contrary to the common expectation for hydroxylamines, 10-aza-9-oxakalkitoxin is not mutagenic.

Reinvestigation of Mycothiazole Reveals the Penta-2,4-dien-1-ol Residue Imparts Picomolar Potency and 8S Configuration.

Reinvestigation of mycothiazole (1) revealed picomolar potency (IC50 = 0.00016, 0.00027, 0.00035 µM) against pancreatic, (PANC-1), liver (HepG2), and colon (HCT-116) tumor cell lines. Reevaluation of 1 provided [α]D data indicating Vanuatu specimens of C. mycofijiensis contain the 8S enantiomer of 1 and not the 8R configuration previously reported. Semisynthesis provided 8-O-acetylmycothiazole (2), 8-oxomycothiazole (8), mycothiazole nitrosobenzene derivatives (MND1, MND2: 9a, 9b), and MND3 (10) with IC50 = 0.00129, >1.0, >1.0, >1.0, >1.0 µM, respectively, against PANC-1 cell lines. These results highlight the significance of the penta-2,4-dien-1-ol residue as a key structural feature of 1 required for its cytotoxicty against tumor cell lines.

A Versatile Chemoenzymatic Synthesis for the Discovery of Potent Cryptophycin Analogs.

The cryptophycins are a family of macrocyclic depsipeptide natural products that display exceptionally potent antiproliferative activity against drug-resistant cancers. Unique challenges facing the synthesis and derivatization of this complex group of molecules motivated us to investigate a chemoenzymatic synthesis designed to access new analogs for biological evaluation. The cryptophycin thioesterase (CrpTE) and the cryptophycin epoxidase (CrpE) are a versatile set of enzymes that catalyze macrocyclization and epoxidation of over 20 natural cryptophycin metabolites. Thus, we envisioned a drug development strategy involving their use as standalone biocatalysts for production of unnatural derivatives. Herein, we developed a scalable synthesis of 12 new unit A-B-C-D linear chain elongation intermediates containing heterocyclic aromatic groups as alternatives to the native unit A benzyl group. N-Acetyl cysteamine activated forms of each intermediate were assessed for conversion to macrocyclic products using wild type CrpTE, which demonstrated the exceptional flexibility of this enzyme. Semipreparative scale reactions were conducted for isolation and structural characterization of new cryptophycins. Each was then evaluated as a substrate for CrpE P450 and its ability to generate the epoxidized products from these substrates that possess altered electronics at the unit A styrenyl double bond position. Finally, biological evaluation of the new cryptophycins revealed a des-ß-epoxy analog with low picomolar potency, previously limited to cryptophycins bearing epoxide functionality.

Computationally Assisted Discovery and Assignment of a Highly Strained and PANC-1 Selective Alkaloid from Alaska's Deep Ocean.

We report here the orchestration of molecular ion networking and a set of computationally assisted structural elucidation approaches in the discovery of a new class of pyrroloiminoquinone alkaloids that possess selective bioactivity against pancreatic cancer cell lines. Aleutianamine represents the first in a new class of pyrroloiminoquinone alkaloids possessing a highly strained multibridged ring system, discovered from Latrunculia ( Latrunculia) austini Samaai, Kelly & Gibbons, 2006 (class Demospongiae, order Poecilosclerida, family Latrunculiidae) recovered during a NOAA deep-water exploration of the Aleutian Islands. The molecule was identified with the guidance of mass spectrometry, nuclear magnetic resonance, and molecular ion networking (MoIN) analysis. The structure of aleutianamine was determined using extensive spectroscopic analysis in conjunction with computationally assisted quantifiable structure elucidation tools. Aleutianamine exhibited potent and selective cytotoxicity toward solid tumor cell lines including pancreatic cancer (PANC-1) with an IC50 of 25 nM and colon cancer (HCT-116) with an IC50 of 1 µM, and represents a potent and selective candidate for advanced preclinical studies.

Highly Convergent Total Synthesis and Assignment of Absolute Configuration of Majusculamide D, a Potent and Selective Cytotoxic Metabolite from Moorea sp.

The total synthesis of majusculamide D (MJS-D) is described, a lipopentapeptide originally isolated from Lyngbya majuscula and reisolated from a Moorea sp. MJS-D possesses selective and potent cancer cell toxicity. A scalable and convergent strategy with a minimal number of purifications produced significant quantities of MJM-D for in vivo evaluations. The absolute configuration of the 1,3-dimethyl-octanamide motif was determined by synthesis of this fragment via ZACA chemistry.

Exploration of the carmaphycins as payloads in antibody drug conjugate anticancer agents.

Antibody-drug conjugates (ADCs) represent a new dimension of anticancer chemotherapeutics, with warheads to date generally involving either antitubulin or DNA-directed agents to achieve low-to sub-nanomolar potency. However, other potent cytotoxins working by different pharmacological mechanisms are under investigation, such as α,ß-epoxyketone based proteasome inhibitors. These proteasome active agents are an emerging class of anticancer drug that possesses ultra-potent cytotoxicity to some cancer cell lines. The carmaphycins are representatives of this latter class that we isolated and characterized from a marine cyanobacterium, and these as well as several synthetic analogues exhibit this level of potency. In the current work, we investigated the use of these highly potent cytotoxic compounds as warheads in the design of novel ADCs. We designed and synthesized a library of carmaphycin B analogues that contain amine handles, enabling their attachment to an antibody linker. The basicity of these incorporated amine handles was shown to strongly affect their cytotoxic properties. Linear amines resulted in the greatest reduction in cytotoxicity whereas less basic aromatic amines retained potent activity as demonstrated by a 4-sulfonylaniline derivative. These investigations resulted in identifying the P2 residue in the carmaphycins as the most suitable site for linker attachment point, and hence, we synthesized a highly potent analogue of carmaphycin B that contained a 4-sulfonylaniline handle as an attachment point for the linker antibody.

Computationally Assisted Assignment of the Kadsuraols, a Class of Chemopreventive Agents for the Control of Liver Cancer.

Kadsuraols A-C (1-3), which are tetrahydrocyclobutaphenanthrofuranone-type lignans with a new carbon skeleton comprising a four-membered ring across C-1'-C-8, have been isolated from the roots of Kadsura longipedunculata. Their structures and absolute configurations were unambiguously determined using nuclear magnetic resonance, X-ray diffraction crystallography, DP4+ calculations, and computed and experimental electronic circular dichroism spectra. Kadsuraol C (3) exhibited hepatoprotective activity against N-acetyl- p-aminophenol (APAP)-induced toxicity. The compounds showed no cytotoxicity at 10 µM in a zone assay.

The potential of achiral sponge-derived and synthetic bromoindoles as selective cytotoxins against PANC-1 tumor cells.

Our quest to isolate and characterize natural products with in vitro solid tumor selectivity is driven by access to repositories of Indo-Pacific sponge extracts. In this project an extract of a species of Haplosclerida sponge obtained from the US NCI Natural Products Repository displayed, by in vitro disk diffusion assay (DDA) and IC50 determinations, selective cytotoxicity with modest potency to a human pancreatic cancer cell line (PANC-1) relative to the human lymphoblast leukemia cell line (CCRF-CEM). Two brominated indoles, the known 6-bromo conicamin (1) and the new derivative, 6-Br-8-keto-conicamin A (2), were identified and 2 (IC50 1.5 µM for the natural product vs 4.1 µM for the synthetic material) was determined to be responsible for the cytotoxic activity of the extract against the PANC-1 tumor cell line. The new natural product and ten additional analogs were prepared for further SAR testing.

Investigation of the Physical and Bioactive Properties of Bromo- and Iodo-Containing Sponge-Derived Compounds Possessing an Oxyphenylethanamine Core.

This research set out to identify compounds from marine sponges that can act as bacterial virulence blockers. Extracts from a total of 80 sponges collected from throughout Indonesia were screened in a high-throughput NF-κB-based screen that identifies compounds capable of inhibiting the bacterial type III secretion system (T3SS) in Yersinia pseudotuberculosis. An extract that was shown to inhibit T3SS-driven NF-κB expression was obtained from an Iotrochota cf. iota sponge and was the source of seven new bromo- and iodo-containing compounds, all of which contain a 2-(4-oxyphenyl)ethan-1-amine core. Five were determined to be new compounds and named enisorines A-E (1-5). The remaining two were determined to be new hemibastadinol analogues named (+)-1-O-methylhemibastadinol 2 (6) and (+)-1-O-methylhemibastadinol 4 (7). All seven compounds inhibited T3SS-dependent YopE secretion and did not affect the growth or metabolic activity of Y. pseudotuberculosis. The most potent inhibitors of T3SS activity were enisorine C (3), enisorine E (5), and (+)-1-O-methylhemibastadinol 2 (6), all of which inhibited YopE secretion by >50% at 30 µM.

Another Look at Pyrroloiminoquinone Alkaloids-Perspectives on Their Therapeutic Potential from Known Structures and Semisynthetic Analogues.

This study began with the goal of identifying constituents from Zyzzya fuliginosa extracts that showed selectivity in our primary cytotoxicity screen against the PANC-1 tumor cell line. During the course of this project, which focused on six Z. fuliginosa samples collected from various regions of the Indo-Pacific, known compounds were obtained consisting of nine makaluvamine and three damirone analogues. Four new acetylated derivatives were also prepared. High-accuracy electrospray ionization mass spectrometry (HAESI-MS) m/z ions produced through MS² runs were obtained and interpreted to provide a rapid way for dereplicating isomers containing a pyrrolo[4,3,2-de]quinoline core. In vitro human pancreas/duct epithelioid carcinoma (PANC-1) cell line IC50 data was obtained for 16 compounds and two therapeutic standards. These results along with data gleaned from the literature provided useful structure activity relationship conclusions. Three structural motifs proved to be important in maximizing potency against PANC-1: (i) conjugation within the core of the ABC-ring; (ii) the presence of a positive charge in the C-ring; and (iii) inclusion of a 4-ethyl phenol or 4-ethyl phenol acetate substituent off the B-ring. Two compounds, makaluvamine J (9) and 15-O-acetyl makaluvamine J (15), contained all three of these frameworks and exhibited the best potency with IC50 values of 54 nM and 81 nM, respectively. These two most potent analogs were then tested against the OVCAR-5 cell line and the presence of the acetyl group increased the potency 14-fold from that of 9 whose IC50 = 120 nM vs. that of 15 having IC50 = 8.6 nM.

Integrating Molecular Networking and Biological Assays To Target the Isolation of a Cytotoxic Cyclic Octapeptide, Samoamide A, from an American Samoan Marine Cyanobacterium.

Integrating LC-MS/MS molecular networking and bioassay-guided fractionation enabled the targeted isolation of a new and bioactive cyclic octapeptide, samoamide A (1), from a sample of cf. Symploca sp. collected in American Samoa. The structure of 1 was established by detailed 1D and 2D NMR experiments, HRESIMS data, and chemical degradation/chromatographic (e.g., Marfey's analysis) studies. Pure compound 1 was shown to have in vitro cytotoxic activity against several human cancer cell lines in both traditional cell culture and zone inhibition bioassays. Although there was no particular selectivity between the cell lines tested for samoamide A, the most potent activity was observed against H460 human non-small-cell lung cancer cells (IC50 = 1.1 µM). Molecular modeling studies suggested that one possible mechanism of action for 1 is the inhibition of the enzyme dipeptidyl peptidase (CD26, DPP4) at a reported allosteric binding site, which could lead to many downstream pharmacological effects. However, this interaction was moderate when tested in vitro at up to 10 µM and only resulted in about 16% peptidase inhibition. Combining bioassay screening with the cheminformatics strategy of LC-MS/MS molecular networking as a discovery tool expedited the targeted isolation of a natural product possessing both a novel chemical structure and a desired biological activity.

Cytotoxic Phyllactone Analogs from the Marine Sponge Phyllospongia papyrecea.

BACKGROUND: A growing evidence indicates that marine sponge Phyllospongia sp. is one of rich sources of 20, 24-bishomoscalarane sesterterpenes with potent biological activities. In order to search more bioactive 20, 24-bishomoscalarane sesterterpenes for new drug discovery, chemical investigation was carried out on an Indonesian marine sponge P. papyrecea. METHODS: Bioassay-guided fractionation was carried out on its dichloromethane extract. And nine compounds were purified and isolated using HPLC. Their chemical structures were determined by a combination of spectroscopic and spectrometric data, including 1D-, 2D-NMR and HRESI-MS. Their cytotoxic activities were performed on three human tumor cell lines A549, MCF-7 and HeLa using the CCK-8 method. RESULTS: One new 20, 24-bishomoscalarane sesterterpene, phyllactone H (9), was isolated and elucidated together with phyllactones A-B (1-2) and D-G (3-6), 12α, 24-dihydroxy-20, 24-dimethyl-15, 17- scalaradien-25, 24-olides (7-8). Compounds 1 and 2, 3 and 4, 5 and 6, 7 and 8 were C-24 anomers and inseparable mixtures, respectively. The 1H and 13C-NMR data for 7/8 were firstly reported in this paper. CONCLUSION: Compounds 1-9 possessed in vitro moderate cytotoxicities against A549, MCF-7 and HeLa cells with IC50 values of less than 25 µM.

Cytotoxic halogenated monoterpenes from Plocamium cartilagineum.

As a result of our efforts to identify bioactive agents from marine algae, we have isolated and identified one new halogenated monoterpene 1 [(-)-(5E,7Z)-348-trichloro-7-dichloromethyl-3-methyl-157-octatriene] in addition to three known compounds (2, 3 and 4) from the red alga Plocamium cartilagineum collected by hand from the eastern coast of South Africa. Compound 1 was found to be active as a cytotoxic agent in human lung cancer (NCI-H460) and mouse neuro-2a cell lines (IC50 4 µg/mL). Two of these compounds (3 and 4) were found to have cytotoxic activity in other cell line assays, especially against human leukaemia and human colon cancers (IC50 1.3 µg/mL). None of these metabolites were active as sodium channel blockers or activators. All structures were determined by spectroscopic methods (UV, IR, LRMS, HRMS, 1D NMR and 2D NMR). 1D and 2D NOE experiments were carried out on these compounds to confirm the geometry of the double bonds.

Mycotoxin verrucarin A inhibits proliferation and induces apoptosis in prostate cancer cells by inhibiting prosurvival Akt/NF-kB/mTOR signaling.

Trichothecenes are powerful mycotoxins that inhibit protein synthesis and induce ribotoxic stress response in mammalian cells. Verrucarin A (VC-A) is a Type D macrocyclic mycotoxin which inhibits cell proliferation and induces apoptosis in cancer cells. However, the antitumor activity of VC-A for prostate cancer cells has not been investigated. The objective of the present study was to determine the anticancer activity and its mechanism of action in hormone-responsive (LNCaP) and hormone-refractory (PC-3) carcinoma of the prostate (CaP) cell lines. VC-A strongly inhibited the proliferation and induced cell cycle arrest in G2/M phase associated with the inhibition of cell cycle regulatory proteins cyclin D, cyclin E, cyclin-dependent kinases (cdks) cdk2, cdk4, cdk6 and cdk inhibitors WAF1/21 and KIP1/27. VC-A also induced apoptosis in CaP cells as characterized by the cleavage of poly (ADP-ribose) polymerase (PARP-1), procaspases-3, -8 and -9 and the inhibition of Bcl-2 family proteins that regulate apoptosis (Bcl-2, Bcl-xL, Bax, Bak and Bad). In addition, VC-A also down-regulated the expression of prosurvival phospho-AKT (p-AKT), nuclear factor kappa B (NF-kB) (p65) and phospho-mammalian target of rapamycin (p-mTOR) signaling proteins. Taken together, these results demonstrated strong antiproliferative and apoptosis-inducing activity of verrucarin A against CaP cells through cell cycle arrest and inhibition of the prosurvival (antiapoptotic) AKT/NF-kB/mTOR signaling pathway.

The inhibition of cell proliferation and induction of apoptosis in pancreatic ductal adenocarcinoma cells by verrucarin A, a macrocyclic trichothecene, is associated with the inhibition of Akt/NF-кB/mTOR prosurvival signaling.

Pancreatic ductal adenocarcinoma (PDA) remains one of the most difficult to treat of all malignancies. Multimodality regimens provide only short-term symptomatic improvement with minor impact on survival, underscoring the urgent need for novel therapeutics and treatment strategies for PDA. Trichothecenes are powerful mycotoxins that inhibit protein synthesis and induce ribotoxic stress response in mammalian cells. Verrucarin A (VC-A) is a Type D macrocyclic mycotoxin which inhibited cell proliferation and induced apoptosis in breast cancer cells. However, the antitumor activity of VC-A for PDA cells has not been investigated. Here we show potent antitumor activity and the mechanism of action of VC-A in PDA cell lines. VC-A strongly inhibited the proliferation and arrested cells in the S phase of the cell cycle. The blocking of cell cycle progression by VC-A was associated with the inhibition of cell cycle regulatory proteins cyclin D1, cyclin E, cyclin-dependent kinases (cdks) cdk2, cdk4 and cdk inhibitor WAF1/21. VC-A induced apoptosis in PDA cells as indicated by the increased Annexin V FITC-binding, cleavage of poly(ADP-ribose) polymerase­1 (PARP-1) and procaspases-3, -8 and -9. VC-A also induced mitochondrial depolarization and release of cytochrome c and it inhibited Bcl-2 family proteins that regulate apoptosis (Bcl-2, Bcl-xL, Bax and Bad). In addition, VC-A reduced the levels of inhibitors of apoptosis survivin and c-IAP-2. Finally, VC-A downregulated the expression of prosurvival phospho-Akt (p-Akt), nuclear factor κB (NF-κB) (p65) and mammalian target of rapamycin (p-mTOR) signaling proteins and their downstream mediators. Together, these results demonstrated strong antiproliferative and apoptosis-inducing activity of verrucarin A for PDA cells through cell cycle arrest and inhibition of the prosurvival (antiapoptotic) AKT/NF-κB/mTOR signaling.

An efficient and cost-effective approach to kahalalide F N-terminal modifications using a nuisance algal bloom of Bryopsis pennata.

BACKGROUND: Kahalalide F (KF) and its isomer iso-kahalalide F (isoKF), both of which can be isolated from the mollusk Elysia rufescens and its diet alga Bryopsis pennata, are potent cytotoxic agents that have advanced through five clinical trials. Due to a short half-life, narrow spectrum of activity, and a modest response in patients, further efforts to modify the molecule are required to address its limitations. In addition, due to the high cost in producing KF analogues using solid phase peptide synthesis (SPPS), a degradation and reconstruction approach was employed using natural KF from a seasonal algal bloom to generate KF analogues. METHODS: N-protected KF was carefully hydrolyzed at the amide linkage between l-Thr12 and d-Val13 using dilute HCl. The synthesis of the C-terminal fragment began with the formation of hexanoic succinimide ester, followed by a reaction with dipeptides. The final coupling reaction was performed between the semisynthesized Fmoc-KF hydrolysis product and the C-terminal fragment, followed by the deprotection of the Fmoc group. RESULTS: Six KF analogues with an addition of an amino acid residue on the N-terminal chain, d-Val14-isoKF (2), Val13-Val14-isoKF (3), d-Leu14-isoKF (4), d-Pro14-isoKF (5), d-Phe14-isoKF (6), and 3,4-2F-d-Phe14-isoKF (7) were prepared using semisynthesis at the exposed N-terminal chain. CONCLUSIONS: The overall yield of the medication was 45%. This approach is economical, efficient and amendable to large-scale production while eliminated a nuisance algal bloom. GENERAL SIGNIFICANCE: B. pennata blooms are capable of producing KF in good yields. The semisynthesis from the natural product produced N-terminal modifications for the construction of inexpensive semisynthetic KF libraries.

Kalkitoxin inhibits angiogenesis, disrupts cellular hypoxic signaling, and blocks mitochondrial electron transport in tumor cells.

The biologically active lipopeptide kalkitoxin was previously isolated from the marine cyanobacterium Moorea producens (Lyngbya majuscula). Kalkitoxin exhibited N-methyl-D-aspartate (NMDA)-mediated neurotoxicity and acted as an inhibitory ligand for voltage-sensitive sodium channels in cultured rat cerebellar granule neurons. Subsequent studies revealed that kalkitoxin generated a delayed form of colon tumor cell cytotoxicity in 7-day clonogenic cell survival assays. Cell line- and exposure time-dependent cytostatic/cytotoxic effects were previously observed with mitochondria-targeted inhibitors of hypoxia-inducible factor-1 (HIF-1). The transcription factor HIF-1 functions as a key regulator of oxygen homeostasis. Therefore, we investigated the ability of kalkitoxin to inhibit hypoxic signaling in human tumor cell lines. Kalkitoxin potently and selectively inhibited hypoxia-induced activation of HIF-1 in T47D breast tumor cells (IC50 5.6 nM). Mechanistic studies revealed that kalkitoxin inhibits HIF-1 activation by suppressing mitochondrial oxygen consumption at electron transport chain (ETC) complex I (NADH-ubiquinone oxidoreductase). Further studies indicate that kalkitoxin targets tumor angiogenesis by blocking the induction of angiogenic factors (i.e., VEGF) in tumor cells.

Biosynthetic products from a nearshore-derived gram-negative bacterium enable reassessment of the kailuin depsipeptides.

Sampling of California nearshore sediments resulted in the isolation of a Gram-negative bacterium, Photobacterium halotolerans, capable of producing unusual biosynthetic products. Liquid culture in artificial seawater-based media provided cyclic depsipeptides including four known compounds, kailuins B-E (2-5), and two new analogues, kailuins G and H (7 and 8). The structures of the new and known compounds were confirmed through extensive spectroscopic and Marfey's analyses. During the course of these studies, a correction was made to the previously reported double-bond geometry of kailuin D (4). Additionally, through the application of a combination of derivatization with Mosher's reagent and extensive (13)C NMR shift analysis, the previously unassigned chiral center at position C-3 of the ß-acyloxy group of all compounds was determined. To evaluate bioactivity and structure-activity relationships, the kailuin core (13) and kailuin lactam (14) were prepared by chiral synthesis using an Fmoc solid-phase peptide strategy followed by solution-phase cyclization. All isolated compounds and synthetic cores were assayed for solid tumor cell cytotoxicity and showed only minimal activity, contrary to other published reports. Additional phenotypic screenings were done on 4 and 5, with little evidence of activity.