Andrew M. White

Choose Your Weaponry: Selective Storage of a Single Toxic Compound, Latrunculin A, by Closely Related Nudibranch Molluscs

Natural products play an invaluable role as a starting point in the drug discovery process, and plants and animals use many interesting biologically active natural products as a chemical defense mechanism against predators. Among marine organisms, many nudibranch gastropods are known to derive defensive metabolites from the sponges they eat. Here we investigated the putative sequestration of the toxic compound latrunculin A—a 16-membered macrolide that prevents actin polymerization within cellular processes—which has been identified from sponge sources, by five closely related nudibranch molluscs of the genus Chromodoris. Only latrunculin A was present in the rim of the mantle of these species, where storage reservoirs containing secondary metabolites are located, whilst a variety of secondary metabolites were found in their viscera. The species studied thus selectively accumulate latrunculin A in the part of the mantle that is more exposed to potential predators. This study also demonstrates that latrunculin-containing sponges are not their sole food source. Latrunculin A was found to be several times more potent than other compounds present in these species of nudibranchs when tested by in vitro and in vivo toxicity assays. Anti-feedant assays also indicated that latrunculin A was unpalatable to rock pool shrimps, in a dose-dependent manner. These findings led us to propose that this group of nudibranchs has evolved means both to protect themselves from the toxicity of latrunculin A, and to accumulate this compound in the mantle rim for defensive purposes. The precise mechanism by which the nudibranchs sequester such a potent compound from sponges without disrupting their own key physiological processes is unclear, but this work paves the way for future studies in this direction. Finally, the possible occurrence of both visual and chemosensory Müllerian mimicry in the studied species is discussed.

Discovery and optimization of peptide macrocycles

ABSTRACT Introduction: Macrocyclic peptides are generally more resistant to proteolysis and often have higher potency than linear peptides and so they are excellent leads in drug design. Their study is significant because they offer potential as a new generation of drugs that are potent and specific, and thus might have fewer side effects than traditional small molecule drugs. Areas covered: This article covers macrocyclic drug leads based on nature-derived cyclic peptides as well as synthetic cyclic peptides and close derivatives. The natural peptides include cyclotides, sunflower-derived peptides, theta-defensins and orbitides. Technologies to make engineered cyclic peptides covered here include cyclization via amino acid linkers, CLIPS, templates, and stapled peptides. Expert opinion: Macrocyclic peptides are promising drug leads and several are in clinical trials. The authors believe they offer key advantages over traditional small molecule drugs, as well as some advantages over protein-based ‘biologics’ such as antibodies or growth factors. These include the ability to penetrate cells and attack intracellular targets such as protein-protein interactions as well as to hit extracellular targets. Some macrocyclic peptides such as cyclotides offer the potential for production in plants, thus reducing manufacture costs and potentially increasing opportunities for their distribution to developing countries at low cost.

Rearranged Diterpenes and Norditerpenes from Three Australian Goniobranchus Mollusks

Three new norditerpenes (1, 6, and 7) and four diterpenes (2-5) with extensively rearranged carbon skeletons have been characterized from Australian nudibranchs. The relative configuration of the cyclopropyl-containing verrielactone (1) from Goniobranchus verrieri was suggested by spectroscopic analysis at 500 MHz informed by a combination of molecular modeling and DFT calculations. The nudibranchs G. splendidus and G. cf. splendidus provided 2-7, for which the structures and stereochemistry were deduced by 2D NMR studies at either 500 or 700 MHz. Each of the seven terpenoids exhibited a carbon skeleton modified from one of the tetrahydroaplysulphurin, spongionellin, or gracilane series of terpenes. A biosynthetic pathway to terpenes 1-7 from spongialactone is proposed.

New Cytotoxic Norditerpenes from the Australian Nudibranchs Goniobranchus Splendidus and Goniobranchus Daphne

This study reports the isolation and characterisation of six new metabolites with ‘gracilin’-type carbon skeletons and of aplytandiene-3 from the Australian nudibranch Goniobranchus splendidus. The structure of gracilin G is revised, and the C-6 configuration deduced by comparison of calculated 3JC/H values with values measured using the EXSIDE pulse sequence. A lactone isolated from Goniobranchus daphne contains a rearranged spongionellin-type skeleton. Screening of selected metabolites revealed significant cytotoxicity against a HeLa S3 cell line by five of the new terpenes.

Antimalarial Isocyano and Isothiocyanato Sesquiterpenes with Tri- and Bicyclic Skeletons from the Nudibranch Phyllidia ocellata

Five new isocyano/isothiocyanato sesquiterpenes (1-5) with tri- or bicyclic carbon skeletons have been characterized from Australian specimens of the nudibranch Phyllidia ocellata. Spectroscopic analyses at 900 MHz were informed by DFT calculations. The 1S, 5S, 8R configuration of 2-isocyanoclovene (1) was determined by X-ray crystallographic analysis of formamide 6. A biosynthetic pathway to clovanes 1 and 2 from epicaryolane precursors is proposed. Isocyanides 1, 2, and 4 showed activity against Plasmodium falciparum (IC50 0.26-0.30 μM), while isothiocyanate 3 and formamide 6 had IC50 values of >10 μM.

Chemoecological studies on marine natural products: terpene chemistry from marine mollusks

Abstract Some species of nudibranchs (Mollusca) protect themselves from predatory attacks by storing defensive terpene chemicals acquired from dietary sponges (Porifera) in specialized body parts called MDFs (mantle dermal formations), often advertising their unpalatability to potential predators by means of bright coloration patterns. Consequently, the survival of these trophic specialist species is closely related to the possibility of obtaining the defensive tools from sponges that live in their immediate vicinity; therefore, it is important to determine as precisely as possible the chemical composition of nudibranch extracts prior to any ecological studies addressing issues that involve their alimentary behavior and their defensive strategies, including the significance of their color patterns. Some of our recent studies on the chemical composition of terpene extracts from nudibranchs belonging to the genera Chromodoris and Hypselodoris are summarized. We also report the development of a method to assay extracts and purified metabolites for their feeding deterrent activity against co-occurring generalist predators. In a recent chemoecological study, showing that repugnant terpene chemicals are accumulated at extremely high concentrations in exposed parts of the nudibranchs’ bodies, the feeding deterrence assays were carried out on the generalist marine shrimp Palaemon elegans, very common in the Mediterranean. We have modified this assay for use with the Australian shrimp species P. serenus, and confirmed the ecological validity of the assay by analysis of extracts from species of sponges and mollusks that live in the same habitat as P. serenus. The deterrent properties of haliclonacyclamine alkaloids isolated from the sponge Haliclona sp. were demonstrated, with the alkaloid mixture demonstrating palatability deterrence at concentrations as low as 0.05 mg/mL, and complete deterrence at 0.75 mg/mL. In contrast, the diterpene thuridillin metabolites from the sacoglossan mollusk Thuridilla splendens did not deter feeding by P. serenus.

Distribution of Defensive Metabolites in Nudibranch Molluscs

Many plants and animals store toxic or unpalatable compounds in tissues that are easily encountered by predators during attack. Defensive compounds can be produced de novo, or obtained from dietary sources and stored directly without selection or modification, or can be selectively sequestered or biotransformed. Storage strategies should be optimized to produce effective defence mechanisms but also prevent autotoxicity of the host. Nudibranch molluscs utilize a diverse range of chemical defences, and we investigated the accumulation and distribution of defensive secondary metabolites in body tissues of 19 species of Chromodorididae nudibranchs. We report different patterns of distribution across tissues, where: 1) the mantle had more or different (but structurally related) compounds than the viscera; 2) all compounds in the mantle were also in the viscera; and 3) the mantle had fewer compounds than the viscera. We found no further examples of species that selectively store a single compound, previously reported in Chromodoris species. Consistent with other studies, we found high concentrations of metabolites in mantle rim tissues compared to the viscera. Using bioassays, compounds in the mantle were more toxic than compounds found in the viscera for Glossodoris vespa Rudman, 1990 and Ceratosoma brevicaudatum Abraham, 1876. In G. vespa, compounds in the mantle were also more unpalatable to palaemonid shrimp than compounds found in the viscera. This indicates that these species may modify compounds to increase bioactivity for defensive purposes and/or selectively store more toxic compounds. We highlight clear differences in the storage of sequestered chemical defences, which may have important implications for species to employ effective defences against a range of predators.

Toxicity and taste: unequal chemical defences in a mimicry ring

Mimicry of warning signals is common, and can be mutualistic when mimetic species harbour equal levels of defence (Müllerian), or parasitic when mimics are undefended but still gain protection from their resemblance to the model (Batesian). However, whether chemically defended mimics should be similar in terms of toxicity (i.e. causing damage to the consumer) and/or unpalatability (i.e. distasteful to consumer) is unclear and in many studies remains undifferentiated. In this study, we investigated the evolution of visual signals and chemical defences in a putative mimicry ring of nudibranch molluscs. First, we demonstrated that the appearance of a group of red spotted nudibranchs molluscs was similar from the perspective of potential fish predators using visual modelling and pattern analysis. Second, using phylogenetic reconstruction, we demonstrated that this colour pattern has evolved multiple times in distantly related individuals. Third, we showed that these nudibranchs contained different chemical profiles used for defensive purposes. Finally, we demonstrated that although levels of distastefulness towards Palaemon shrimp remained relatively constant between species, toxicity levels towards brine shrimp varied significantly. We highlight the need to disentangle toxicity and taste when considering chemical defences in aposematic and mimetic species, and discuss the implications for aposematic and mimicry signal evolution.

Development of Novel Melanocortin Receptor Agonists Based on the Cyclic Peptide Framework of Sunflower Trypsin Inhibitor-1

Ultrastable cyclic peptide frameworks offer great potential for drug design due to their improved bioavailability compared to their linear analogues. Using the sunflower trypsin inhibitor-1 (SFTI-1) peptide scaffold in combination with systematic N-methylation of the grafted pharmacophore led to the identification of novel subtype selective melanocortin receptor (MCR) agonists. Multiple bicyclic peptides were synthesized and tested toward their activity at MC1R and MC3-5R. Double N-methylated compound 18 showed a p Ki of 8.73 ± 0.08 ( Ki = 1.92 ± 0.34 nM) and a pEC50 of 9.13 ± 0.04 (EC50 = 0.75 ± 0.08 nM) at the human MC1R and was over 100 times more selective for MC1R. Nuclear magnetic resonance structural analysis of 18 emphasized the role of peptide bond N-methylation in shaping the conformation of the grafted pharmacophore. More broadly, this study highlights the potential of cyclic peptide scaffolds for epitope grafting in combination with N-methylation to introduce receptor subtype selectivity in the context of peptide-based drug discovery.

Catalyst-Controlled Stereoselective Synthesis Secures the Structure of the Antimalarial Isocyanoterpene Pustulosaisonitrile-1

Three new isocyanoditerpenes (5-7) have been characterized from Australian specimens of the nudibranch Phyllidiella pustulosa. The planar structure and (3R,6S,7R) absolute configuration of pustulosaisonitrile-1 were deduced by spectroscopic analyses at 900 MHz informed by molecular modeling, DFT calculations, and computational NMR chemical shift predictions and by comparison of experimental electronic circular dichroism (ECD) data with TDDFT-ECD calculations for the truncated model compound 8. A catalyst-controlled enantio- and diastereoselective total synthesis of the two most likely diastereomeric candidates for the structure of 5 solidified its (3R,6S,7R,10S,11R,14R) absolute configuration. Three individual enantioselective methods provided stereochemical control at key positions, permitting an unambiguous final structural assignment. Isocyanide 5 and synthetic diastereomers 5a and 5c showed activity against Plasmodium falciparum malaria parasites (IC50 ∼1 μM).