The Asymmetric Total Synthesis and Configuration Confirmation of Aplysiaenal and Nhatrangin A, Truncated Derivatives of Aplysiatoxin and Oscillatoxin.

Asymmetric total syntheses of aplysiaenal (1) and nhatrangin A (2), truncated derivatives of the aplysiatoxin/oscillatoxin family of marine natural products, from configurationally defined intermediates are described. NMR spectra of our synthesized nhatrangin A did not match with either those obtained from authentic samples of the natural product or material obtained via two other total syntheses, but were similar to that obtained from a sample obtained in a third total synthesis. By independently synthesizing the fragments used in its total syntheses, we were able to confirm the configuration of nhatrangin A and clarified that the discrepancy in the spectroscopic data is due to salt formation of the carboxylic acid moiety.

Structure Elucidation of Two Intriguing Neo-Debromoaplysiatoxin Derivatives from Marine Cyanobacterium Lyngbya sp. Showing Strong Inhibition of Kv1.5 Potassium Channel and Differential Cytotoxicity.

Two aplysiatoxin derivatives, neo-debromoaplysiatoxin I (1) and neo-debromoaplysiatoxin J (2), were isolated from marine cyanobacterium Lyngbya sp. collected from the South China Sea. Their structures including absolute configurations were assigned by spectroscopic analysis, in combination with GIAO NMR shift calculation and DP4+ analysis. Structures of neo-debromoaplysiatoxin I and neo-debromoaplysiatoxin J contained a decahydro-5H-pyrano [2,3,4-de] chromen-5-one 6/6/6 ring skeleton and an intriguing peroxide bridge group, respectively, which are unprecedented structure scaffold and motif in aplysiatoxins. Two compounds displayed comparable inhibitory activities against Kv1.5 K+ channel with IC50 values of 2.59 ± 0.37 µM (1) and 1.64 ± 0.15 µM (2); however, they presented differential cytotoxic effects. It is worth noting that neo-debromoaplysiatoxin J, containing a peroxide bridge, showed remarkable cytotoxicity against four cancer cell lines including SW480, SGC7901, LoVo and PC-9 compared to the human normal cell line.

Functions, Structures, and Engineering of the Teleocidin Biosynthetic Enzymes.

Teleocidins are natural products belonging to the indole alkaloid family and show potent protein kinase C activation activity. The structural feature of teleocidins is an indole-fused nine-membered lactam ring structure. Due to their unique structures and strong biological activities, many total synthesis and biosynthetic studies of teleocidins have been performed. Teleocidin biosynthesis involves interesting enzymatic reactions that are challenging in organic synthesis, including oxidative intramolecular C-N bond-forming reactions, regio- and stereo-selective reverse prenylation reactions, and methylation-triggered terpene cyclization. This review summarizes the recent research on functional and structural analyses, as well as enzyme engineering, of teleocidin biosynthetic enzymes.

Anti-Infective Secondary Metabolites of the Marine Cyanobacterium Lyngbya Morphotype between 1979 and 2022.

Cyanobacteria ascribed to the genus Lyngbya (Family Oscillatoriaceae) represent a potential therapeutic gold mine of chemically and biologically diverse natural products that exhibit a wide array of biological properties. Phylogenetic analyses have established the Lyngbya 'morpho-type' as a highly polyphyletic group and have resulted in taxonomic revision and description of an additional six new cyanobacterial genera in the same family to date. Among the most prolific marine cyanobacterial producers of biologically active compounds are the species Moorena producens (previously L. majuscula, then Moorea producens), M. bouillonii (previously L. bouillonii), and L. confervoides. Over the years, compounding evidence from in vitro and in vivo studies in support of the significant pharmaceutical potential of 'Lyngbya'-derived natural products has made the Lyngbya morphotype a significant target for biomedical research and novel drug leads development. This comprehensive review covers compounds with reported anti-infective activities through 2022 from the Lyngbya morphotype, including new genera arising from recent phylogenetic re-classification. So far, 72 anti-infective secondary metabolites have been isolated from various Dapis, Lyngbya, Moorea, and Okeania species. These compounds showed significant antibacterial, antiparasitic, antifungal, antiviral and molluscicidal effects. Herein, a comprehensive literature review covering the natural source, chemical structure, and biological/pharmacological properties will be presented.

A simplified analog of debromoaplysiatoxin lacking the B-ring of spiroketal moiety retains protein kinase C-binding and antiproliferative activities.

A simplified analog (3) of aplysiatoxin was synthesized. Compound 3 has only one tetrahydropyran ring at positions 3-7, the A-ring of the spiroketal moiety, which is the conformation-controlling unit for the macrolactone ring. Nuclear magnetic resonance (NMR) analysis and density functional theory (DFT) calculations indicated that 3 existed as an equilibrium mixture of two conformers arising from inversion of the chair conformation of the 2,6-trans-tetrahydropyran ring. The des-B-ring analog 3 binds protein kinase C isozymes and exhibits antiproliferative activity toward human cancer cell lines, comparable to 18-deoxy-aplog-1 with a spiroketal moiety.

A review and assessment of cyanobacterial toxins as cardiovascular health hazards.

Eutrophicated waters frequently support bloom-forming cyanobacteria, many of which produce potent cyanobacterial toxins (cyanotoxins). Cyanotoxins can cause adverse health effects in a wide range of organisms where the toxins may target the liver, other internal organs, mucous surfaces and the skin and nervous system. This review surveyed more than 100 studies concerning the cardiovascular toxicity of cyanotoxins and related topics. Over 60 studies have described various negative effects on the cardiovascular system by seven major types of cyanotoxins, i.e. the microcystin (MC), nodularin (NOD), cylindrospermopsin (CYN), anatoxin (ATX), guanitoxin (GNTX), saxitoxin (STX) and lyngbyatoxin (LTX) groups. Much of the research was done on rodents and fish using high, acutely toxin concentrations and unnatural exposure routes (such as intraperitoneal injection), and it is thus concluded that the emphasis in future studies should be on oral, chronic exposure of mammalian species at environmentally relevant concentrations. It is also suggested that future in vivo studies are conducted in parallel with studies on cells and tissues. In the light of the presented evidence, it is likely that cyanotoxins do not constitute a major risk to cardiovascular health under ordinary conditions met in everyday life. The risk of illnesses in other organs, in particular the liver, is higher under the same exposure conditions. However, adverse cardiovascular effects can be expected due to indirect effects arising from damage in other organs. In addition to risks related to extraordinary concentrations of the cyanotoxins and atypical exposure routes, chronic exposure together with co-existing diseases could make some of the cyanotoxins more dangerous to cardiovascular health.

Design, synthesis, and biological activity of a synthetically accessible analog of aplysiatoxin with an (R)-(-)-carvone-based conformation-controlling unit.

Simplified analogs of aplysiatoxin (ATX) such as 10-Me-aplog-1 exhibit potent antiproliferative activity toward human cancer cell lines by activating protein kinase C (PKC). However, the synthesis of 10-Me-aplog-1 involved a 23-step longest linear sequence (LLS). Therefore, we have been working toward the development of a more synthetically accessible analog of ATX. In this study, we designed a new analog of ATX wherein a cyclic ketal moiety derived from (R)-(-)-carvone replaced the spiroketal moiety in 18-deoxy-aplog-1. The new analog's synthesis proceeded in an 8-step LLS. Although the configuration at position 3 of the cyclic ketal in the (R)-(-)-carvone-based analog was opposite to those of ATX and 18-deoxy-aplog-1, the antiproliferative activity toward human cancer cell lines of the carvone-based analog was comparable with that of 18-deoxy-aplog-1. The obtained results indicate the potential of the carvone-based analog as a basis for discovering PKC-targeting molecules requiring a decreased number of synthetic steps.

Teleocidin-producing genotype of Streptomyces clavuligerus ATCC 27064.

Streptomyces clavuligerus is an industrially important producer of clavulanic acid (CA), a ß-lactamase inhibitor which is used together with amoxicillin in one of the most widely prescribed antibacterial medicines, the co-amoxiclav. In a mid-eighties ATCC vial of S. clavuligerus ATCC 27064 culture, we have found a new genotype, which was apparently lost from the subsequent ATCC collection stocks, and has remained obscure to the scientific community. Most importantly, this genotype harbors teleocidin (lyngbyatoxin) biosynthetic genes, which are located on an enigmatic 138 kb chromosomal region and support accumulation of significant amounts of these highly toxic, tumor-promoting secondary metabolites in cultures of S. clavuligerus. While this genomic region is completely absent from all published sequences for S. clavuligerus ATCC strain, at least one of the industrial strains for commercial production of CA, originating from ATCC 27064, retained the genetic potential for production of teleocidins. The origin of teleocidin biosynthetic cluster can now be traced back to early S. clavuligerus stocks at the ATCC. Our work provides a genome sequence and a deposited monoisolate of this genotype. Given the scale of industrial use of S. clavuligerus world-wide and toxicity of teleocidins, we also discuss the environmental and safety implications and provide a method of abolishing teleocidin production without affecting productivity of CA. KEY POINTS: • Early stocks of S. clavuligerus ATCC 27064 produce toxic teleocidins • Teleocidin biosynthetic genes were found within a distinct S. clavuligerus genotype • The genotype has been passed on to some industrial clavulanic acid producer strains.

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.

Tailoring Enzyme Stringency Masks the Multispecificity of a Lyngbyatoxin (Indolactam Alkaloid) Nonribosomal Peptide Synthetase.

Indolactam alkaloids are activators of protein kinase C (PKC) and are of pharmacological interest for the treatment of pathologies involving PKC dysregulation. The marine cyanobacterial nonribosomal peptide synthetase (NRPS) pathway for lyngbyatoxin biosynthesis, which we previously expressed in E. coli, was studied for its amenability towards the biosynthesis of indolactam variants. Modification of culture conditions for our E. coli heterologous expression host and analysis of pathway products suggested the native lyngbyatoxin pathway NRPS does possess a degree of relaxed specificity. Site-directed mutagenesis of two positions within the adenylation domain (A-domain) substrate-binding pocket was performed, resulting in an alteration of substrate preference between valine, isoleucine, and leucine. We observed relative congruence of in vitro substrate activation by the LtxA NRPS to in vivo product formation. While there was a preference for isoleucine over leucine, the substitution of alternative tailoring domains may unveil the true in vivo effects of the mutations introduced herein.

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.

Enzymatic reactions in teleocidin B biosynthesis.

The teleocidin B family members are terpene indole compounds isolated from Streptomyces bacteria, and they strongly activate protein kinase C (PKC). Their unique structures have attracted many researchers in the natural product chemistry and pharmacology fields, and numerous isolation and bioactivity studies have been conducted. The accumulated information has facilitated the identification of the enzymatic reactions in teleocidin biosynthesis, and new developments in structural biology have strongly aided efforts to clarify the finer points of these reactions. This review describes the recent biochemical and structural biological studies to reveal their reaction mechanisms, with a primary focus on the terpene cyclization triggered by the C-N bond formation by P450 oxygenase (TleB), the prenyltransferase (TleC), and the methyltransferase (TleD). This new knowledge will benefit future engineering studies to create unnatural PKC activators.

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.

Enzymatic Formation of Indolactam Scaffold by C-N Bond-Forming Cytochrome P450 Oxidases in Teleocidin Biosynthesis.

Teleocidins are potent protein kinase C activators, and possess a unique indole-fused nine-membered lactam structure. Teleocidin biosynthesis starts from the formation of a dipeptide by non-ribosomal peptide synthetase (NRPS), followed by oxidative C-N bond formation by a cytochrome P450 oxidase, reverse-prenylation by a prenyltransferase, and methylation-initiated terpene cyclization by a C-methyltransferase. This minireview focuses on recent research progress toward the elucidation of the molecular basis for the remarkable P450-catalyzed intramolecular C-N bond-forming reaction, which is challenging in synthetic chemistry, to generate the indolactam scaffold. In addition, precursor-directed biosynthesis with the promiscuous P450 enzymes led to the formation of a series of unnatural and novel molecular scaffolds, including a sulfur-substituted indolactam with a different conformation from that of indolactam V.

Nematicidal activity of teleocidin B4 isolated from Streptomyces sp. against pine wood nematode, Bursaphelenchus xylophilus.

BACKGROUND: Pine wilt disease (PWD) is a plant disease that causes serious damage to pine trees. PWD occurs when the host plant is infected with pinewood nematode (PWN), Bursaphelenchus xylophilus. In this study, a compound with nematicidal activity was isolated from actinomycetes and its efficacy was investigated in vitro. RESULT: We screened and selected Streptomyces sp. 680560, which had nematicidal activity against B. xylophilus. Based on 16S rRNA sequence analysis, it showed 99.93% similarity with Streptomyces blastmyceticus NRRB-5480T . Furthermore, the active compound was isolated and identified as teleocidin B4. Teleocidin B4 at concentrations ranging from 6.25 to 100 µM had low nematicidal activity after 24 and 36 h against adult and stage juveniles (J2) of B. xylophilus, but after 48 h nematicidal activity exceeded 95%. The rate of inhibition of egg hatching for Teleocidin B4 6.25, 12.5, 25, 50, and 100 µM was confirmed to be dose-dependently inhibited after 48 h of treatment. Teleocidin B4 is not only toxic to hatched B. xylophilus, but also affects egg hatching. CONCLUSION: This study was carried out to isolate actinomycete metabolites from pine tree endophytes from various natural environments for control of PWD. A compound with nematicidal activity was isolated from a selected strain and its structure was identified as teleocidin B4. The nematicidal effect of the isolated active substance, teleocidin B4, was confirmed. This is the first report of the effect of teleocidin B4 on B. xylophilus, suggesting its possibility as a PWD control agent. © 2020 Society of Chemical Industry.

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.

Chemical and Biological Study of Novel Aplysiatoxin Derivatives from the Marine Cyanobacterium Lyngbya sp.

Since 1970s, aplysiatoxins (ATXs), a class of biologically active dermatoxins, were identified from the marine mollusk Stylocheilus longicauda, whilst further research indicated that ATXs were originally metabolized by cyanobacteria. So far, there have been 45 aplysiatoxin derivatives discovered from marine cyanobacteria with various geographies. Recently, we isolated two neo-debromoaplysiatoxins, neo-debromoaplysiatoxin G (1) and neo-debromoaplysiatoxin H (2) from the cyanobacterium Lyngbya sp. collected from the South China Sea. The freeze-dried cyanobacterium was extracted with liquid-liquid extraction of organic solvents, and then was subjected to multiple chromatographies to yield neo-debromoaplysiatoxin G (1) (3.6 mg) and neo-debromoaplysiatoxin H (2) (4.3 mg). They were elucidated with spectroscopic methods. Moreover, the brine shrimp toxicity of the aplysiatoxin derivatives representing differential structural classifications indicated that the debromoaplysiatoxin was the most toxic compound (half inhibitory concentration (IC50) value = 0.34 ± 0.036 µM). While neo-aplysiatoxins (neo-ATXs) did not exhibit apparent brine shrimp toxicity, but showed potent blocking action against potassium channel Kv1.5, likewise, compounds 1 and 2 with IC50 values of 1.79 ± 0.22 µM and 1.46 ± 0.14 µM, respectively. Therefore, much of the current knowledge suggests the ATXs with different structure modifications may modulate multiple cellular signaling processes in animal systems leading to the harmful effects on public health.

Neo-Aplysiatoxin A Isolated from Okinawan Cyanobacterium Moorea Producens.

A new aplysiatoxin derivative, neo-aplysiatoxin A (1), along with seven known compounds, neo-debromoaplysiatoxin A (2), dolastatin 3 (3), lyngbic acid (4), malyngamide M (5), hermitamide A (6), (-)-loliolide (7), and (+)-epiloliolide (8), was isolated from the Okinawan cyanobacterium Moorea producens. Their structures were elucidated on the basis of spectroscopic data, including high-resolution mass spectrometry and nuclear magnetic resonance. The compounds were evaluated for cytotoxic and diatom growth inhibition activities.

Neo-debromoaplysiatoxin C, with new structural rearrangement, derived from debromoaplysiatoxin.

Neo-debromoaplysiatoxin C (1), a new member of the aplysiatoxin family, was isolated from the marine cyanobacterium Lyngbya sp. The structure of 1 was elucidated based on spectroscopic data, and its stereochemistry was determined from NOESY spectrum and biosynthetic considerations. This new compound presents an intriguing 10-membered lactone ring skeleton derived from debromoaplysiatoxin by structural rearrangement, which is the first example in the aplysiatoxin family. Its biological properties were evaluated for cytotoxicity, PKCδ activation and inhibitory effects on potassium channel.