Effect of phenolic-hydroxy-group incorporation on the biological activity of a simplified aplysiatoxin analog with an (R)-(-)-carvone-based core.

We synthesized a phenolic hydroxy group-bearing version (1) of a simplified analog of aplysiatoxin comprising a carvone-based conformation-controlling unit. Thereafter, we evaluated its antiproliferative activity against human cancer cell lines and its binding affinity to protein kinase C (PKC) isozymes. The antiproliferative activity and PKC-binding ability increased with the introduction of the phenolic hydroxy group. The results of molecular dynamics simulations and subsequent relative binding free-energy calculations conducted using an alchemical transformation procedure showed that the phenolic hydroxy group in 1 could form a hydrogen bond with a phospholipid and the PKC. The former hydrogen bonding formation facilitated the partitioning of the compound from water to the phospholipid membrane and the latter compensated for the loss of hydrogen bond with the phospholipid upon binding to the PKC. This information may facilitate the development of rational design methods for PKC ligands with additional hydrogen bonding groups.

Bursatella leachii Purple Ink Secretion Concentrate Exerts Cytotoxic Properties against Human Hepatocarcinoma Cell Line (HepG2): In Vitro and In Silico Studies.

Liver cancer is a leading cause of cancer death globally. Marine mollusc-derived drugs have gained attention as potential natural-based anti-cancer agents to overcome the side effects caused by conventional chemotherapeutic drugs during cancer therapy. Using liquid chromatography-mass spectrometry, the main biomolecules in the purple ink secretion released by the sea hare, named Bursatella leachii (B. leachii), were identified as hectochlorin, malyngamide X, malyngolide S, bursatellin and lyngbyatoxin A. The cytotoxic effects of B. leachii ink concentrate against human hepatocarcinoma (HepG2) cells were determined to be dose- and time-dependent, and further exploration of the underlying mechanisms causing the programmed cell death (apoptosis) were performed. The expression of cleaved-caspase-8 and cleaved-caspase-3, key cysteine-aspartic proteases involved in the initiation and completion of the apoptosis process, appeared after HepG2 cell exposure to the B. leachii ink concentrate. The gene expression levels of pro-apoptotic BAX, TP53 and Cyclin D1 were increased after treatment with the B. leachii ink concentrate. Applying in silico approaches, the high scores predicted that bioactivities for the five compounds were protease and kinase inhibitors. The ADME and cytochrome profiles for the compounds were also predicted. Altogether, the B. leachii ink concentrate has high pro-apoptotic potentials, suggesting it as a promising safe natural product-based drug for the treatment of liver cancer.

Absolute Structure Determination and Kv1.5 Ion Channel Inhibition Activities of New Debromoaplysiatoxin Analogues.

Potassium channel Kv1.5 has been considered a key target for new treatments of atrial tachyarrhythmias, with few side effects. Four new debromoaplysiatoxin analogues with a 6/6/12 fused ring system were isolated from marine cyanobacterium Lyngbya sp. Their planar structures were elucidated by HRESIMS, 1D and 2D NMR. The absolute configuration of oscillatoxin J (1) was determined by single-crystal X-ray diffraction, and the absolute configurations of oscillatoxin K (2), oscillatoxin L (3) and oscillatoxin M (4) were confirmed on the basis of GIAO NMR shift calculation followed by DP4 analysis. The current study confirmed the absolute configuration of the pivotal chiral positions (7S, 9S, 10S, 11R, 12S, 15S, 29R and 30R) at traditional ATXs with 6/12/6 tricyclic ring system. Compound 1, 2 and 4 exhibited blocking activities against Kv1.5 with IC50 values of 2.61 ± 0.91 µM, 3.86 ± 1.03 µM and 3.79 ± 1.01 µM, respectively. However, compound 3 exhibited a minimum effect on Kv1.5 at 10 µM. Furthermore, all of these new debromoaplysiatoxin analogs displayed no apparent activity in a brine shrimp toxicity assay.

Effects of side chain length of 10-methyl-aplog-1, a simplified analog of debromoaplysiatoxin, on PKC binding, anti-proliferative, and pro-inflammatory activities.

10-Methyl-aplog-1 (1), a simplified analog of debromoaplysiatoxin, exhibits a high binding affinity for protein kinase C (PKC) isozymes and potent antiproliferative activity against several cancer cells with few adverse effects. A recent study has suggested that its phenol group in the side chain is involved in hydrogen bonding and CH/π interactions with the binding cleft-forming loops in the PKCδ-C1B domain. To clarify the effects of the side chain length on these interactions, four analogs of 1 with various lengths of side chains (2-5) were prepared. The maximal PKC binding affinity and antiproliferative activity were observed in 1. Remarkably, the introduction of a bromine atom into the phenol group of 2 increased not only these activities but also proinflammatory activity. These results indicated that 1 has the optimal side chain length as an anticancer seed. This conclusion was supported by docking simulations of 1-5 to the PKCδ-C1B domain.

Synthesis and biological activities of simplified aplysiatoxin analogs focused on the CH/π interaction.

Debromoaplysiatoxin (DAT) is a potent protein kinase C (PKC) activator with tumor-promoting and pro-inflammatory activities. Irie and colleagues have found that 10-methyl-aplog-1 (1), a simplified analog of DAT, has strong anti-proliferative activity against several cancer cell lines with few adverse effects. Therefore, 1 is a potential lead compound for cancer therapy. We synthesized a new derivative 2 which has a naphthalene ring at the side chain terminal position instead of a benzene ring, to increase CH/π interactions with Pro-241 of the PKCδ-C1B domain. Based on the synthetic route of 1, 2 was convergently synthesized in 26 linear steps from 6-hydroxy-1-naphthoic acid with an overall yield of 0.18%. Although the anti-proliferative activity of 2 was more potent than that of 1, the binding potency of 2 to the PKCδ-C1B domain did not exceed that of 1. Molecular dynamics simulation indicated the capability of 2 to simultaneously form hydrogen bonds and CH/π interactions with the PKCδ-C1B domain. Focusing on the hydrogen bonds, their geometry in the binding modes involving the CH/π interactions seemed to be sub-optimal, which may explain the slightly lower affinity of 2 compared to 1. This study could be of help in optimizing such interactions and synthesizing a promising lead cancer compound.

Two New Neo-debromoaplysiatoxins-A Pair of Stereoisomers Exhibiting Potent Kv1.5 Ion Channel Inhibition Activities.

A pair of stereoisomers possessing novel structures with 6/6/5 fused-ring systems, neo-debromoaplysiatoxin E (1) and neo-debromoaplysiatoxin F (2), were isolated from the marine cyanobacterium Lyngbya sp. Their structures were elucidated using various spectroscopic techniques including high resolution electrospray ionization mass spectroscopy (HRESIMS) and nuclear magnetic resonance (NMR). The absolute stereochemistry was determined by calculated electronic circular dichroism (ECD) and gauge-independent atomic orbital (GIAO) NMR shift calculation followed by DP4+ analysis. Significantly, this is the first report on aplysiatoxin derivatives with different absolute configurations at C9-C12 (1: 9S, 10R, 11S, 12S; 2: 9R, 10S, 11R, 12R). Compounds 1 and 2 exhibited potent blocking activities against Kv1.5 with IC50 values of 1.22 ± 0.22 µM and 2.85 ± 0.29 µM, respectively.

Identification of protein kinase C isozymes involved in the anti-proliferative and pro-apoptotic activities of 10-Methyl-aplog-1, a simplified analog of debromoaplysiatoxin, in several cancer cell lines.

10-Me-aplog-1 is a simplified analog of the tumor-promoting compound debromoaplysiatoxin (DAT) and a unique protein kinase C (PKC) activator with limited tumor-promoting and pro-inflammatory activities. 10-Me-aplog-1 inhibits the growth of several cancer cell lines, but the inhibitory mechanism involving PKC isozymes remains unclear. We quantified the amount of PKC isozymes in nine human cancer cell lines that differ in 10-Me-aplog-1 sensitivity. PKCα and δ were the predominant isozymes expressed in all cell lines, but there was no significant correlation between expression levels and anti-proliferative activity. Knocking down PKCα, and/or PKCδ in the three aplog-sensitive cell lines indicated their involvement in the anti-proliferative and pro-apoptotic activities of 10-Me-aplog-1. This finding suggests that PKCα and/or PKCδ activation could be effective for treating certain cancers. Since the mechanism underlying 10-Me-aplog-1's anti-proliferative activities resembles that of DAT, 10-Me-aplog-1 may be regarded as a special key derived from pleiotropic DAT as a bunch of keys.

Loss of the Phenolic Hydroxyl Group and Aromaticity from the Side Chain of Anti-Proliferative 10-Methyl-aplog-1, a Simplified Analog of Aplysiatoxin, Enhances Its Tumor-Promoting and Proinflammatory Activities.

Aplysiatoxin (ATX) is a protein kinase C (PKC) activator with potent tumor-promoting activity. In contrast, 10-methyl-aplog-1 (1), a simplified analog of ATX, was anti-proliferative towards several cancer cell lines without significant tumor-promoting and proinflammatory activities. To determine the effects of the phenolic group on the biological activities of 1, we synthesized new derivatives (2, 3) that lack the phenolic hydroxyl group and/or the aromatic ring. Compound 2, like 1, showed potent anti-proliferative activity against several cancer cell lines, but little with respect to tumor-promoting and proinflammatory activities. In contrast, 3 exhibited weaker growth inhibitory activity, and promoted inflammation and tumorigenesis. The binding affinity of 3 for PKCδ, which is involved in growth inhibition and apoptosis, was several times lower than those of 1 and 2, possibly due to the absence of the hydrogen bond and CH/π interaction between its side chain and either Met-239 or Pro-241 in the PKCδ-C1B domain. These results suggest that both the aromatic ring and phenolic hydroxyl group can suppress the proinflammatory and tumor-promoting activities of 1 and, therefore, at least the aromatic ring in the side chain of 1 is indispensable for developing anti-cancer leads with potent anti-proliferative activity and limited side effects. In accordance with the binding affinity, the concentration of 3 necessary to induce PKCδ-GFP translocation to the plasma membrane and perinuclear regions in HEK293 cells was higher than that of 1 and 2. However, the translocation profiles for PKCδ-GFP due to induction by 1-3 were similar.

Structural optimization of 10-methyl-aplog-1, a simplified analog of debromoaplysiatoxin, as an anticancer lead.

Aplog-1 is a simplified analog of debromoaplysiatoxin (DAT) with potent tumor-promoting and proinflammatory activities. Aplog-1 and DAT exhibited anti-proliferative activities against several human cancer cell lines, whereas aplog-1 did not have tumor-promoting nor proinflammatory activities. We have recently found 10-methyl-aplog-1 (1) to have strong anti-proliferative activity compared with aplog-1. To further investigate the structural factors involved in the tumor-promoting, proinflammatory, and anti-proliferative activities, two dimethyl derivatives of aplog-1 (2, 3) were synthesized, where two methyl groups were installed at positions 4 and 10 or 10 and 12. 10,12-Dimethyl-aplog-1 (2) had stronger inhibitory effects on the growth of several human cancer cell lines than 1 and DAT, but exhibited no tumor-promoting and proinflammatory activities. In contrast, 4,10-dimethyl-aplog-1 (3) displayed weak tumor-promoting and proinflammatory activities along with anti-proliferative activity similar to that of 1 and DAT. Compound 2 would be the optimized seed for anticancer drugs among the simplified analogs of DAT.

Studies toward the total synthesis of Itralamide B and biological evaluation of its structural analogs.

Itralamides A and B were isolated from the lipophilic extract of Lyngbya majuscula collected from the eastern Caribbean. Itralamide B (1) showed cytotoxic activity towards human embryonic kidney cells (HEK293, IC50 = 6 µM). Preliminary studies disapproved the proposed stereochemistry of itralamide. In this paper, we will provide a full account of the total synthesis of four stereoisomers of itralamide B and the results derived from biological tests of these structural congeners.

Synthesis and biological activities of the amide derivative of aplog-1, a simplified analog of aplysiatoxin with anti-proliferative and cytotoxic activities.

Aplog-1 is a simplified analog of the tumor-promoting aplysiatoxin with anti-proliferative and cytotoxic activities against several cancer cell lines. Our recent findings have suggested that protein kinase Cδ (PKCδ) could be one of the target proteins of aplog-1. In this study, we synthesized amide-aplog-1 (3), in which the C-1 ester group was replaced with an amide group, to improve chemical stability in vivo. Unfortunately, 3 exhibited seventy-fold weaker binding affinity to the C1B domain of PKCδ than that of aplog-1, and negligible anti-proliferative and cytotoxic activities even at 10(-4) M. A conformational analysis and density functional theory calculations indicated that the stable conformation of 3 differed from that of aplog-1. Since 27-methyl and 27-methoxy derivatives (1, 2) without the ability to bind to PKC isozymes exhibited marked anti-proliferative and cytotoxic activities at 10(-4) M, 3 may be an inactive control to identify the target proteins of aplogs.

A new lyngbyatoxin from the Hawaiian cyanobacterium Moorea producens.

Lyngbyatoxin A from the marine cyanobacterium Moorea producens (formerly Lyngbya majuscula) is known as the causative agent of "swimmer's itch" with its highly inflammatory effect. A new toxic compound was isolated along with lyngbyatoxin A from an ethyl acetate extract of M. producens collected from Hawaii. Analyses of HR-ESI-MS and NMR spectroscopies revealed the isolated compound had the same planar structure with that of lyngbyatoxin A. The results of optical rotation and CD spectra indicated that the compound was a new lyngbyatoxin A derivative, 12-epi-lyngbyatoxin A (1). While 12-epi-lyngbyatoxin A showed comparable toxicities with lyngbyatoxin A in cytotoxicity and crustacean lethality tests, it showed more than 100 times lower affinity for protein kinase Cδ (PKCδ) using the PKCδ-C1B peptide when compared to lyngbyatoxin A.

Synthesis and biological activities of simplified analogs of the natural PKC ligands, bryostatin-1 and aplysiatoxin.

Protein kinase C (PKC) isozymes play central roles in signal transduction on the cell surface and could serve as promising therapeutic targets of intractable diseases like cancer, Alzheimer's disease, and acquired immunodeficiency syndrome (AIDS). Although natural PKC ligands like phorbol esters, ingenol esters, and teleocidins have the potential to become therapeutic leads, most of them are potent tumor promoters in mouse skin. By contrast, bryostatin-1 (bryo-1) isolated from marine bryozoan is a potent PKC activator with little tumor-promoting activity. Numerous investigations have suggested bryo-1 to be a promising therapeutic candidate for the above intractable diseases. However, there is a supply problem of bryo-1 both from natural sources and by organic synthesis. Recent approaches on the synthesis of bryo-1 have focused on its simplification, without decreasing the ability to activate PKC isozymes, to develop new medicinal leads. Another approach is to use the skeleton of natural PKC ligands to develop bryo-1 surrogates. We have recently identified 10-methyl-aplog-1 (26), a simplified analog of tumor-promoting aplysiatoxin (ATX), as a possible therapeutic lead for cancer. This review summarizes recent investigations on the simplification of natural PKC ligands, bryo-1 and ATX, to develop potential medicinal leads.

Anti-Chikungunya viral activities of aplysiatoxin-related compounds from the marine cyanobacterium Trichodesmium erythraeum.

Tropical filamentous marine cyanobacteria have emerged as a viable source of novel bioactive natural products for drug discovery and development. In the present study, aplysiatoxin (1), debromoaplysiatoxin (2) and anhydrodebromoaplysiatoxin (3), as well as two new analogues, 3-methoxyaplysiatoxin (4) and 3-methoxydebromoaplysiatoxin (5), are reported for the first time from the marine cyanobacterium Trichodesmium erythraeum. The identification of the bloom-forming cyanobacterial strain was confirmed based on phylogenetic analysis of its 16S rRNA sequences. Structural determination of the new analogues was achieved by extensive NMR spectroscopic analysis and comparison with NMR spectral data of known compounds. In addition, the antiviral activities of these marine toxins were assessed using Chikungunya virus (CHIKV)-infected cells. Post-treatment experiments using the debrominated analogues, namely compounds 2, 3 and 5, displayed dose-dependent inhibition of CHIKV when tested at concentrations ranging from 0.1 µM to 10.0 µM. Furthermore, debromoaplysiatoxin (2) and 3-methoxydebromoaplysiatoxin (5) exhibited significant anti-CHIKV activities with EC50 values of 1.3 µM and 2.7 µM, respectively, and selectivity indices of 10.9 and 9.2, respectively.

Bisebromoamide, an extract from Lyngbya species, induces apoptosis through ERK and mTOR inhibitions in renal cancer cells.

Advanced renal cell carcinoma (RCC) remains an incurable disease, and newer anticancer drugs are needed. Bisebromoamide, a novel cytotoxic peptide, was isolated from the marine cyanobacterium Lyngbya species at our laboratory in 2009. This compound specifically inhibited the phosphorylation of ERK in platelet-derived growth factor-activated normal rat kidney cells. The aim of this study was to evaluate the effect and elucidate the potential mechanism of Bisebromoamide actions on human RCC cells. Two renal cancer cell lines, 769-P and 786-O, were used. The effects of Bisebromoamide were analyzed employing assays for water-soluble Tetrazolium-1 salts. Apoptosis was determined by flow cytometric TUNEL analysis. Cell-cycle distributions were analyzed by flow cytometry using BrdU/propidium iodide (PI) staining. Kinases of the phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of Rapamycin (mTOR) pathway and Raf/MEK/ERK pathway were analyzed by Western blotting. After Bisebromoamide treatment for 48 and 72 h, cell viability was significantly decreased in both cell lines at 1 and 10 µmol/L. After treatment with 1 µmol/L Bisebromoamide for 72 h, apoptosis and the increased percentage of cells in the sub-G1 phase were observed in both cell lines. Bisebromoamide inhibited the phosphorylation of ERK and Akt in both cell lines tested. Similar effects were demonstrated for phosphorylation of mTOR and p70 S6. Bisebromoamide is a promising potential agent against RCC due to its ability to inhibit both the Raf/MEK/ERK and PI3K/Akt/mTOR pathways.

Apratoxin H and apratoxin A sulfoxide from the Red Sea cyanobacterium Moorea producens.

Cultivation of the marine cyanobacterium Moorea producens, collected from the Nabq Mangroves in the Gulf of Aqaba (Red Sea), led to the isolation of new apratoxin analogues apratoxin H (1) and apratoxin A sulfoxide (2), together with the known apratoxins A-C, lyngbyabellin B, and hectochlorin. The absolute configuration of these new potent cytotoxins was determined by chemical degradation, MS, NMR, and CD spectroscopy. Apratoxin H (1) contains pipecolic acid in place of the proline residue present in apratoxin A, expanding the known suite of naturally occurring analogues that display amino acid substitutions within the final module of the apratoxin biosynthetic pathway. The oxidation site of apratoxin A sulfoxide (2) was deduced from MS fragmentation patterns and IR data, and 2 could not be generated experimentally by oxidation of apratoxin A. The cytotoxicity of 1 and 2 to human NCI-H460 lung cancer cells (IC50 = 3.4 and 89.9 nM, respectively) provides further insight into the structure-activity relationships in the apratoxin series. Phylogenetic analysis of the apratoxin-producing cyanobacterial strains belonging to the genus Moorea, coupled with the recently annotated apratoxin biosynthetic pathway, supports the notion that apratoxin production and structural diversity may be specific to their geographical niche.

Structure-activity studies on the side chain of a simplified analog of aplysiatoxin (aplog-1) with anti-proliferative activity.

We have recently developed a simplified analog of aplysiatoxin (aplog-1) as an activator of protein kinase C (PKC) with anti-proliferative activity like bryostain 1. To identify sites in aplog-1 that could be readily modified to optimize therapeutic performance and to develop a molecular probe for examining the analog's mode of action, substituent effects on the phenol ring were systematically examined. Whereas hydrophilic acetamido derivatives were less active than aplog-1 in inhibiting cancer cell growth and binding to PKCδ, introduction of hydrophobic bromine and iodine atoms enhanced both biological activities. The anti-proliferative activity was found to correlate closely with molecular hydrophobicity, and maximal activity was observed at a logP value of 4.0-4.5. On the other hand, an induction test with Epstein-Barr virus early antigen demonstrated that these derivatives have less tumor-promoting activity in vitro than aplog-1 regardless of the hydrophobicity of their substituents. These results would facilitate rapid preparation of molecular probes to examine the mechanism of the unique biological activities of aplog-1.

Biochemical studies of the lagunamides, potent cytotoxic cyclic depsipeptides from the marine cyanobacterium Lyngbya majuscula.

Lagunamides A (1) and B (2) are potent cytotoxic cyclic depsipeptides isolated from the filamentous marine cyanobacterium, Lyngbya majuscula, from Pulau Hantu, Singapore. These compounds are structurally related to the aurilide-class of molecules, which have been reported to possess exquisite antiproliferative activities against cancer cells. The present study presents preliminary findings on the selectivity of lagunamides against various cancer cell lines as well as their mechanism of action by studying their effects on programmed cell death or apoptosis. Lagunamide A exhibited a selective growth inhibitory activity against a panel of cancer cell lines, including P388, A549, PC3, HCT8, and SK-OV3 cells, with IC50 values ranging from 1.6 nM to 6.4 nM. Morphological studies showed blebbing at the surface of cancer cells as well as cell shrinkage accompanied by loss of contact with the substratum and neighboring cells. Biochemical studies using HCT8 and MCF7 cancer cells suggested that the cytotoxic effect of 1 and 2 might act via induction of mitochondrial mediated apoptosis. Data presented in this study warrants further investigation on the mode of action and underscores the importance of the lagunamides as potential anticancer agents.

Artificial analogs of naturally occurring tumor promoters as biochemical tools and therapeutic leads.

Tumor promoters are non-carcinogenic chemicals that enhance tumor formation when administered repeatedly after a low dose of a carcinogen. Phorbol esters, teleocidins, and aplysiatoxins are typical examples of naturally occurring tumor promoters. All of them share the ability to bind and activate protein kinase C (PKC) despite the differences in their chemical structures. A variety of analogs with unique chemical and biological properties have been developed to analyze the molecular mechanism of tumor promotion through PKC activation. Moreover, coupled with the emerging significance of PKC in the pathological processes of Alzheimer's disease (AD) and acquired immune deficiency syndrome (AIDS) as well as cancer, several efforts have been made recently to generate analogs of tumor promoters with therapeutic potential. This review focuses on artificial analogs of phorbol esters, teleocidins, and aplysiatoxins, and discusses their potential as biochemical tools and therapeutic leads.

Structure-activity studies on the spiroketal moiety of a simplified analogue of debromoaplysiatoxin with antiproliferative activity.

Aplog-1, a simplified analogue of tumor-promoting debromoaplysiatoxin, is antiproliferative but not tumor-promoting. Our recent study has suggested that local hydrophobicity around the spiroketal moiety is a crucial determinant for antiproliferative activity. To further clarify the structural features relevant to the activity, we synthesized two methyl derivatives of aplog-1, where a methyl group was installed at position 4 or 10 of the spiroketal moiety. 10-Methyl-aplog-1 (5) bound to the C1B domains of novel PKCs (δ, η, and θ) with subnanomolar K(i) values, approximately 10-20 times stronger than aplog-1, and markedly inhibited the growth of many human cancer cell lines, while 4-methyl-aplog-1 (4) had levels of activity similar to those of aplog-1. Interestingly, 5 showed little tumor-promoting activity unlike the tumor promoter debromoaplysiatoxin. These results suggest that 5 is a potent PKC ligand without tumor-promoting activity and could be a therapeutic lead for the treatment of cancer, like bryostatins.