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Marine Sponge Cyclic Peptide Theonellamide A Disrupts Lipid Bilayer Integrity without Forming Distinct Membrane Pores
Abstract
Theonellamides (TNMs) are antifungal and cytotoxic bicyclic dodecapeptides derived from the marine sponge Theonella sp. These peptides specifically bind to 3β-hydroxysterols, resulting in 1,3-β-D-glucan overproduction and membrane damage in yeasts. The inclusion of cholesterol or ergosterol in phosphatidylcholine membranes significantly enhanced the membrane affinity of theonellamide A (TNM-A) because of its direct interaction with 3β-hydroxy groups of sterols. To better understand TNM-induced membrane alterations, we investigated the effects of TNM-A on liposome morphology. (31)P nuclear magnetic resonance (NMR) and dynamic light scattering (DLS) measurements revealed that the premixing of TNM-A with lipids induced smaller vesicle formation. When giant unilamellar vesicles were incubated with exogenously added TNM-A, confocal micrographs showed dynamic changes in membrane morphology, which were more frequently observed in cholesterol-containing than sterol-free liposomes. In conjunction with our previous data, these results suggest that the membrane action of TNM-A proceeds in two steps: 1) TNM-A binds to the membrane surface through direct interaction with sterols and 2) accumulated TNM-A modifies the local membrane curvature in a concentration-dependent manner, resulting in dramatic membrane morphological changes and membrane disruption.
... Previous 31 P solid-state NMR studies have revealed that TNM-A disrupts the bilayer integrity of both Chol-containing and Chol-free phospholipid membranes [28]. Thus, the same measurements were carried out with phospholipid membranes containing 25-HC to examine whether a similar membrane disruption occurs. ...
... More importantly, when TNM-A was incorporated into the POPC/25-HC membranes (Fig. 3B), a similar narrow peak, despite a low intensity, was observed around 0 ppm, which indicated that TNM-A was also able to exhibit its membrane disrupting activity in the 25-HC-containing membrane. It could also be explained by a notion that bilayer membrane where 25-HC (or Chol) enhances the local TNM-A concentration undergoes morphological change to raise the curvature as shown by the peak appearing near 0 ppm, which could partly be responsible for membrane disruption and deformation induced by TMN-A [28]. 1 for (B). In both cases, the chemical shift anisotropy (CSA) value obtained as δ (parallel) minus δ (perpendicular) was 40.0 ppm, which was quite similar to that of the POPC-Chol membrane with TNM-A, as shown in the inset spectra [28]. ...
... It could also be explained by a notion that bilayer membrane where 25-HC (or Chol) enhances the local TNM-A concentration undergoes morphological change to raise the curvature as shown by the peak appearing near 0 ppm, which could partly be responsible for membrane disruption and deformation induced by TMN-A [28]. 1 for (B). In both cases, the chemical shift anisotropy (CSA) value obtained as δ (parallel) minus δ (perpendicular) was 40.0 ppm, which was quite similar to that of the POPC-Chol membrane with TNM-A, as shown in the inset spectra [28]. An arrow in panel B depicts that membrane disruption partly occurs by TNM-A in the membrane. ...
Theonellamides (TNMs) are antifungal and cytotoxic bicyclic dodecapeptides isolated from the marine sponge Theonella sp. The inclusion of cholesterol (Chol) or ergosterol in the phosphatidylcholine membrane is known to significantly enhance the membrane affinity for theonellamide A (TNM-A). We have previously revealed that TNM-A stays in a monomeric form in dimethylsulfoxide (DMSO) solvent systems, whereas the peptide forms oligomers in aqueous media. In this study, we utilized 1H NMR chemical shift changes (Δδ1H) in aqueous DMSO solution to evaluate the TNM-A/sterol interaction. Because Chol does not dissolve well in this solvent, we used 25-hydroxycholesterol (25-HC) instead, which turned out to interact with membrane-bound TNM-A in a very similar way to that of Chol. We determined the dissociation constant, KD, by NMR titration experiments and measured the chemical shift changes of TNM-A induced by 25-HC binding in the DMSO solution. Significant changes were observed for several amino acid residues in a certain area of the molecule. The results from the solution NMR experiments, together with previous findings, suggest that the TNM-Chol complex, where the hydrophobic cavity of TNM probably incorporates Chol, becomes less polar by Chol interaction, resulting in a greater accumulation of the peptide in membrane. The deeper penetration of TNM-A into the membrane interior enhances membrane disruption. We also demonstrated that hydroxylated sterols, such as 25-HC that has higher solubility in most NMR solvents than Chol, act as a versatile substitute for sterol and could be used in 1H NMR-based studies of sterol-binding peptides.
... Recently, we have also demonstrated the existence of a direct interaction between sterols in membranes and theonellamide A (TNM-A) (Figure 1), where it recognizes the hydroxyl moiety in the shallow region of the membrane [28]. However, unlike AM3, TNM-A does not seem to form pore structures in membranes composed of binary lipid mixtures, apparent from the very weak dye leakage observed, although the peptide induces disruption of the tight packing of the lipid bilayer [29]. ...
... AM3 and TNM-A are two unique marine natural products that have been shown previously to act via a mechanism that involves direct interaction with membrane lipids resulting to a loss of membrane integrity [19][20][21][22][26][27][28][29]. AM3 shows an absolute dependence in the presence of sterols in order to permeabilize the membrane, which most likely involves the formation of pores. ...
... On the other hand, TNM-A shows a very weak dye leakage potential at all concentrations tested, and these activities were comparable to the previous report using binary lipid mixtures of POPC and cholesterol [28]. These data appear to be consistent with our proposal that TNM-A indeed does not form distinct membrane pores [29]. It has been reported earlier that TNM-A recognizes cholesterol in and preferentially partitions into the l d domain [27], which intuitively should result in faster membrane accumulation compared with lipid mixtures exhibiting no phase separation. ...
Amphidinol 3 (AM3) and theonellamide A (TNM-A) are potent antifungal compounds produced by the dinoflagellate Amphidinium klebsii and the sponge Theonella spp., respectively. Both of these metabolites have been demonstrated to interact with membrane lipids ultimately resulting in a compromised bilayer integrity. In this report, the activity of AM3 and TNM-A in ternary lipid mixtures composed of 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine (POPC):brain sphingomyelin:cholesterol at a mole ratio of 1:1:1 or 3:1:1 exhibiting lipid rafts coexistence is presented. It was found that AM3 has a more extensive membrane permeabilizing activity compared with TNM-A in these membrane mimics, which was almost complete at 15 μM. The extent of their activity nevertheless is similar to the previously reported binary system of POPC and cholesterol, suggesting that phase separation has neither beneficial nor detrimental effects in their ability to disrupt the lipid bilayer.
... Membrane action of theonellamide A proceeds via binding to the membrane surface through direct interaction with sterols and modification of the local membrane curvature in a concentration-dependent manner, resulting in dramatic membrane morphological changes and membrane disruption. Theonellamides represents a new class of sterol-binding molecules that induce membrane damage and activate Rho1-mediated 1,3-beta-D-glucan synthesis [189]. ...
... The mode of action of theonellamides A and F was studied in a serious of studies. These studies demonstrated that theonellamides recognize and interact with 3β-hydroxysterols in lipid membranes and induce major morphological changes in cultured mammalian cells [18] and yeast [19] by activating Rho1-madiated 1,3-β-D-glucan synthesis [20]. Encouraged by the recent findings that the Entotheonella sp. ...
Theonella swinhoei is a fairly common inhabitant of reefs throughout the Indian and Pacific Oceans. Metabolomic analyses of samples of T. swinhoei collected in different depths in the Gulf of Aqaba revealed two chemotypes differing in the profiles of the theonellamides they produce, some of which seem to be unknown. Driven by this finding, we examined a sample of T. swinhoei collected more than 40 years ago in the southern part of the Gulf of Aqaba. Large-scale extract of this sample yielded four theonellamides, the known theopalauamide (4), as the major component, and three new metabolites, theonellamide J (1), 5-cis-Apoa-theopalauamide (2), and theonellamide K (3), as the minor components. The planar structure of these complex cyclic glycopeptides was elucidated by combination of 1D and 2D NMR techniques and HRESIMS. The absolute configuration of the amino acids was established by Marfey’s and advanced Marfey’s methods, and the absolute configuration of its galactose unit using “Tanaka’s method” for monosaccharides. The biological activity of the pure compounds was tested for antibacterial activity and for cytotoxicity to HTC-116 cell line. The compounds presented significant cytotoxicity against the HTC-116 cell line, illuminating the importance of the Apoa subunit for the activity.
... These dodecapeptides show attractive antifungal and cytotoxic bicyclic activities. Theonellamides bind to 3β-hydroxysterols on the yeast membrane surface and cause membrane damage [307]. Aeroplysinin-1 from Aplysina aerophoba demosponge acts as an antibacterial agent against clinically-relevant mircoorganisms [308]. ...
Marine resources have tremendous potential for developing high-value biomaterials. The last decade has seen an increasing number of biomaterials that originate from marine organisms. This field is rapidly evolving. Marine biomaterials experience several periods of discovery and development ranging from coralline bone graft to polysaccharide-based biomaterials. The latter are represented by chitin and chitosan, marine-derived collagen, and composites of different organisms of marine origin. The diversity of marine natural products, their properties and applications are discussed thoroughly in the present review. These materials are easily available and possess excellent biocompatibility, biodegradability and potent bioactive characteristics. Important applications of marine biomaterials include medical applications, antimicrobial agents, drug delivery agents, anticoagulants, rehabilitation of diseases such as cardiovascular diseases, bone diseases and diabetes, as well as comestible, cosmetic and industrial applications.
... [51] TNMs specifically bind to 3β-hydroxysterols, resulting in 1,3-β-D- TNM-A was found to bind to the membrane via shallow insertion, which subsequently modified the local membrane curvature and resulted in disruption of the bilayer integrity (Figure 12b). [52] Furthermore, theopalauamide (28) was found to disrupt the cell membrane via binding to ergosterol and upregulate β-D-glucan synthesis. Also, with the aid of theonellamide A-conjugated gel beads, glutamate dehydrogenase and 17β-hydroxysteroid dehydrogenase IV have been identified as theonellamide-binding proteins. ...
Among peptide‐based drugs, naturally occurring bicyclic compounds have been established as molecules with unique therapeutic potential. The diverse pharmacological activities associated with bicyclic peptides from marine tunicates, sponges, and bacteria render them suitable to be employed as effective surrogate between complex and small therapeutic moieties. Bicyclic peptides possess greater conformational rigidity and higher metabolic stability as compared with linear and monocyclic peptides. The antibody‐like affinity and specificity of bicyclic peptides enable their binding to the challenging drug targets. Bridged macrobicyclic peptides from natural marine resources represent an underexplored class of molecules that provides promising platforms for drug development owing to their biocompatibility, similarity, and chemical diversity to proteins. The present review explores major marine‐derived bicyclic peptides including disulfide‐bridged, histidinotyrosine‐bridged, or histidinoalanine‐bridged macrobicyclic peptides along with their structural characteristics, synthesis, structure–activity relationship, and bioproperties.The comparison of these macrobicyclic congeners with linear/monocyclic peptides along with their therapeutic potential are also briefly discussed. Natural bridged macrobicyclic peptides (BMPs) from marine resources form one of the most promising platforms for drug development owing to their biocompatibility and chemical diversity. This review provides a summary of the structural characteristics, synthesis routes, structure–activity relationships, and bioproperties of natural bioeffective BMPs from marine tunicates, sponges, and bacteria.
... Peptides like theonellamides (TNMs), bicyclic dodecapeptides isolated from Theonella spp. of a marine sponge act by binding to 3β-hydroxysterols in a specific manner and cause overproduction of 1,3-β-D-glucan. They bind to the sterol part of cell membrane and modify the membrane curvature depending upon peptide concentration ultimately leading to damaged membrane structure of fungal yeast cells [51]. Certain peptides even act by dual mechanisms. ...
The current trend of increment in the frequency of antifungal resistance has brought research into an era where new antifungal compounds with novel mechanisms of action are required. Natural antimicrobial peptides, which are ubiquitous components of innate immunity, represent their candidature for novel antifungal peptides. Various antifungal peptides have been isolated from different species ranging from small marine organisms to insects and from various other living species. Based on these peptides, various mimetics of antifungal peptides have also been synthesized using non-natural amino acids. Utilization of these antifungal peptides is somehow limited due to their toxic and unstable nature. This review discusses recent updates and future directions of antifungal peptides, for taking them to the shelf from the bench.
... Membrane action of theonellamide A proceeds via binding to the membrane surface through direct interaction with sterols and modification of the local membrane curvature in a concentration-dependent manner, resulting in dramatic membrane morphological changes and membrane disruption. Theonellamides represents a new class of sterol-binding molecules that induce membrane damage and activate Rho1-mediated 1,3-beta-D-glucan synthesis [189]. ...
Peptides are distinctive biomacromolecules that demonstrate potential cytotoxicity and diversified bioactivities against a variety of microorganisms including bacteria, mycobacteria, and fungi via their unique mechanisms of action. Among broad-ranging pharmacologically active peptides, natural marine-originated thiazole-based oligopeptides possess peculiar structural features along with a wide spectrum of exceptional and potent bioproperties. Because of their complex nature and size divergence, thiazole-based peptides (TBPs) bestow a pivotal chemical platform in drug discovery processes to generate competent scaffolds for regulating allosteric binding sites and peptide–peptide interactions. The present study dissertates on the natural reservoirs and exclusive structural components of marine-originated TBPs, with a special focus on their most pertinent pharmacological profiles, which may impart vital resources for the development of novel peptide-based therapeutic agents.
... In-depth study regarding the interaction of theonellamide A with the membrane revealed that theonellamide A binds to the membrane surface through interaction with sterols and the accumulation of theonellamide A could cause morphological change of the local membrane. With this unique characteristic, theonellamide-based probes would enable visualization of sterol-containing domains in living cells, which could further unveil the dynamic changes in membrane morphology [150]. Yoshida et al. [151] reported the chemogenomic profiling of theonellamide F (Fig. 12), a MNP originally isolated from sponge Theonella sp [152]. ...
Marine natural products represent novel and diverse chemotypes that serve as templates for the discovery and development of therapeutic agents with distinct mechanisms of action. These genetically encoded compounds produced by an evolutionary optimized biosynthetic machinery are usually quite complex and can be difficult to recreate in the laboratory. The isolation from the source organism results in limited amount of material; however, the development of advanced NMR technologies and dereplication strategies has enabled the structure elucidation on small scale. In order to rigorously explore the therapeutic potential of marine natural products and advance them further, the biological characterization has to keep pace with the chemical characterization. The limited marine natural product supply has been a serious challenge for thorough investigation of the biological targets. Several marine drugs have reached the markets or are in clinical trials, where those challenges have been overcome, including through the development of scalable syntheses. However, the identification of mechanisms of action of marine natural products early in the discovery process is potentially game changing, since effectively linking marine natural products to potential therapeutic applications in turn triggers motivation to tackle challenging syntheses and solve the supply problem. An increasing number of sensitive technologies and methods have been developed in recent years, some of which have been successfully applied to marine natural products, increasing the value of these compounds with respect to their biomedical utility. In this review, we discuss advances in overcoming the bottlenecks in marine natural product research, emphasizing on the development and advances of diverse target identification technologies applicable for marine natural product research.
... The structure of theonellamide-A is illustrated in Fig. 7. Theonellamides bind specifically to 3-b-hydroxysterols, resulting in 1,3-b-d-glucan overproduction and membrane damage in yeasts. In this study, they specifically examined (TNM-A) membrane action, that was assessed by 31 P nuclear magnetic resonance (NMR) and confocal microscopy, where they were able to conclude in combination with earlier studies, that TNM-A binding to the membrane is facilitated by the direct interaction of TNM-A with sterol found on the membrane surface, consequently the accumulated TNM-A resulted in disruption of the membrane integrity and distortion in the membrane morphology (Espiritu et al., 2013;Espiritu et al., 2016). ...
Antimicrobial peptides are group of proteins showing broad-spectrum antimicrobial activity that have been known to be powerful agents against a variety of pathogens. This class of compounds contributed to solving the microbial resistance dilemma that limited the use of many potent antimicrobial agents. The marine environment is known to be one of the richest sources for antimicrobial peptides, yet this environment is not fully explored. Hence, the scientific research attention should be directed toward the marine ecosystem as enormous amount of useful discoveries could be brought to the forefront. In the current article, the marine antimicrobial peptides reported from mid 2012 to 2017 have been reviewed.
... In the latter part of this chapter, we discussed the application of solid-state NMR techniques and solution NMR with bicelles to the analysis of membrane interactions of natural products [40,[49][50][51][52][53][54][55][56][57][58][59][60][61][62][63][64][65][66][67]. In particular, NMR-based studies of small molecules with lipids require site-specific isotope-labeled natural products. ...
In this chapter we overview two topics on NMR methodologies for small molecules, mostly natural products; one is about the solution NMR-based methods used for stereochemical determination of natural products, and the other is on the solid-state and other techniques for investigating natural product-membrane interactions. Since important two methods for stereochemical analysis of natural products, namely the J-based configuration analysis (JBCA) and universal NMR database (UDB) methods, were reported in the 1990s, both methods have been widely used in the field of natural products. The newly coming RDC method is not the major method in the field of natural products yet, but will surely be an important tool for the stereochemical correlation between distant stereogenic centers, which could provide invaluable information as to the whole shape of natural products in solution. In the latter part of this chapter, we discuss the application of solid-state and other NMR techniques to membrane interaction analysis of natural products. In particular, we describe three examples of natural products that interact with biological membranes such as amphotericin B, erythromycin A, and theonellamide A. As shown in NMR studies of amphotericin B, natural products often reveal very high affinities for phospholipids and sterols in bilayer membranes. Solid-state NMR, therefore, provides a very promising approach toward the structure study of membrane-active complexes formed by natural products that have high affinity to lipids. In addition, solution NMR techniques can be applied to elucidate the structural features of membrane-bound small molecules such as antibiotic erythromycin A and membrane-disrupting cyclic peptide theonellamide A. Standard 2D ¹H–¹H experiments such as COSY (correlation spectroscopy), NOESY (nuclear Overhauser effect spectroscopy), and DOSY (diffusion-ordered spectroscopy) are often helpful in elucidating the membrane interaction between natural products and lipids.
... Further study of theonellamide-A showed that it binds with 3β-hydroxyl groups of sterols, cholesterol, and ergosterol found in yeast cells and causes membrane disruption and damage in yeasts, which was assessed by solid-state 31P NMR and confocal microscopy. Also, theonellamide-G showed potent antifungal activity against wild and amphotericin B-resistant strains of Candida albicans [75,76]. ...
Nature provides a variety of peptides that are expressed in most living species. Evolutionary pressure and natural selection have created and optimized these peptides to bind to receptors with high affinity. Hence, natural resources provide an abundant chemical space to be explored in peptide-based drug discovery. Marine peptides can be extracted by simple solvent extraction techniques. The advancement of analytical techniques has made it possible to obtain pure peptides from natural resources. Extracted peptides have been evaluated as possible therapeutic agents for a wide range of diseases, including antibacterial, antifungal, antidiabetic and anticancer activity as well as cardiovascular and neurotoxin activity. Although marine resources provide thousands of possible peptides, only a few peptides derived from marine sources have reached the pharmaceutical market. This review focuses on some of the peptides derived from marine sources in the past ten years and gives a brief review of those that are currently in clinical trials or on the market.
Most of the drugs used in medicine have been obtained from natural products or their synthetic derivatives. Bioactive peptides are an important group of natural product sources isolated from marine organisms, plants, animals, and microorganisms. Bioactive peptides have been investigated to promote health because of their high tissue affinity, specificity, and efficiency. Therefore, they possess a key role in cancer, including early diagnosis, prognostic predictors, and treatment. Anticancer effects of peptides have been widely investigated and shown to have many advantages as peptide-based drugs. Many naturally occurring peptides such as plitidepsin, dolastatin 10, didemnin B, and kahalalide F are used in clinical trials for the treatment of various cancers. The structure-activity relationship of these peptides can contribute to the design and development of peptide-based drugs. The aim of this chapter is to review the recent developments in bioactive peptides and their role in cancer therapy.
Although quercetin has a wide range of applications due to its biological properties, its activity may be affected due to its low solubility and its potential for degradation in a physiological environment. The nanoparticulate and microparticulate systems appear as a powerful strategy to enhance its biological activity as well as to contribute to the viability of this compound for pharmacological purposes. Here, we present an innovative and applied research to study the effect of different lipid compositions on the encapsulation of quercetin by using multilamellar vesicles (MLVs). For this purpose, the effects of charge and rigidity in the formulation’s average size, size distribution, charge properties, encapsulation and release efficiencies of this polyphenol were explored. Our results demonstrated that the rigidity imposed by cholesterol increased both the homogeneity of the size distribution and the encapsulation efficiency enabling a significant rate of quercetin release at the system with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine/cholesterol (80:20). The charge modulated both the average size and size distribution as well as resulted in high encapsulation and release efficiencies in the formulation composed by (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol (80:20). To the best of our knowledge, this is the first study concerning charge and rigidity effects on the encapsulation of quercetin in MLVs. Furthermore, the findings presented in this work is a starting point on the use of lipid composition as a modulating agent of important parameters in the development of nano and micro systems for controlled release.
In the present scenario, there is a great demand for targeted treatment to cure various diseases; researchers are searching for an efficient drug to treat pathologies. Aquatic peptides identified from various bio-resources of the aquatic environment such as aquatic plants, microbes and animals are one of the important therapeutic molecules. The aquatic organisms are native to innate immune system that exclusively synthesis numerous immune protein and bioactive peptides to protect the organisms from a variety of life-threatening environmental factors and subsequent pathogenic attack. Aquatic peptides from both freshwater and marine aquatic ecosystems have promising active molecules against pathogenic infection. Each peptide has unique properties to treat the disease. Meanwhile, peptide-based drug development is not an easy task; it requires overcoming various hurdles even in the current advanced techniques available today. This review discusses the potential of aquatic peptides and the limitations in developing them as drug and the current techniques and strategies to achieve a successful peptide-based drug development.
The cell membrane, with high fluidity and alternative curvatures, maintains the robust integrity to distinguish inner and outer space of cells or organelles. Lipids are the main components of the cell membrane, but their functions are largely unknown. Even the visualization of lipids is not straightforward since modification of lipids often hampers its correct physical properties. Many natural products target cell membranes, some of which are used as pharmaceuticals and/or research tools. They show specific recognition on lipids, and thus exhibit desired pharmacological effects and unique biological phenotypes. This review is a catalog of marine natural products that target eukaryotic cell membranes. Chemical structures, biological activities, and molecular mechanisms are summarized. I hope that this review will be helpful for readers to notice the potential of marine natural products in the exploration of the function of lipids and the druggability of eukaryotic cell membranes.
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.
About 70% of the drugs in use are derived from natural products, either used directly or in chemically modified form. Among all possible small molecules (not greater than 5 kDa), only a few of them are biologically active. Natural product libraries may have a higher rate of finding "hits" than synthetic libraries, even with the use of fewer compounds. This is due to the complementarity between the "chemical space" of small molecules and biological macromolecules such as proteins, DNA and RNA, in addition to the three-dimensional complexity of NPs. Chemical probes are molecules which aid in the elucidation of the biological mechanisms behind the action of drugs or drug-like molecules by binding with macromolecular/cellular interaction partners. Probe development and application have been spurred by advancements in photoaffinity label synthesis, affinity chromatography, activity based protein profiling (ABPP) and instrumental methods such as cellular thermal shift assay (CETSA) and advanced/hyphenated mass spectrometry (MS) techniques, as well as genome sequencing and bioengineering technologies. In this review, we restrict ourselves to a survey of natural products (including peptides/mini-proteins and excluding antibodies), which have been applied largely in the last 5 years for the target identification of drugs/drug-like molecules used in research on infectious diseases, and the description of their mechanisms of action.
OSW-1, a unique steroidal saponin isolated from the bulbs of Ornithogalum saundersiae, has potent growth inhibition activity. In this study, we conducted fluorescence measurements and microscopic observations using palmitoyloleoylphosphatidylcholine (POPC)-cholesterol (Chol) bilayers to evaluate the membrane-binding affinity of OSW-1 in comparison with another steroidal saponin, digitonin, and the triterpenoid saponin, soyasaponin Bb(I). The membrane activities of these saponins were evaluated using calcein leakage assays and fitted to the binding isotherm by changing the ratios of saponin:lipids. Digitonin showed the highest binding affinity for the POPC-Chol membrane (Kapp = 0.38 μM-1), and the strongest membrane disruptivity in the bound saponin:lipid ratio at the point of 50% calcein leakage (r50 = 0.47) occurrence. OSW-1 showed slightly lower activity (Kapp = 0.31 μM-1; r50 = 0.78), and the soyasaponin was the lowest in the membrane affinity and the calcein leakage activity (Kapp = 0.017 μM-1; r50= 1.66). The effect of OSW-1 was further assessed using confocal microscopy in an experiment utilizing DiI and rhodamine 6G as the fluorescence probes. The addition of 30 μM OSW-1 induced inward membrane curvature in some giant unilamellar vesicles (GUVs). At the higher OSW-1 concentration (58 μM, r50 = 0.78) where the 50% calcein leakage was observed, the morphology of some GUVs became elongated. With digitonin at the corresponding concentration (35 μM, r50 = 0.47), membrane disruption and formation of large aggregates in aqueous solution were observed, probably due to a detergent-type mechanism. These saponins, including OSW-1, required Chol to exhibit their potent membrane activity, although their mechanisms are thought to be different. At the effective concentration, OSW-1 preferably binds to the bilayers without prominent disruption of vesicles, and exerts its activity through the formation of saponin-Chol complexes, probably resulting in membrane permeabilization.
An orthogonally protected τ-histidinoalanine residue was synthesized via regioselective alkylation of optically pure Boc-l-His-OTCE (TCE = trichloroethyl) with a sulfamidate electrophile derived from Fmoc-d-Ser-OBn. The goal was the synthesis of a non-natural theonellamide, invoking readily accessible variants of the other 10 amino acids. Peptide fragments corresponding to the east and west rings of “theonellamide X″ were synthesized in solution. Each ring was formed independently, providing insights into protecting group limitations, side reactions, and the optimal order of events to approach the formation of the bicyclic system. Ultimately, an undecapeptide was prepared, with the eastern ring formed. The twelfth amino acid, an l-α-aminoadipic acid building block, was prepared and preliminary investigations into its attachment to the undecapeptide are reported.
Covering: up to 2019
Nature furnishes bioactive compounds (natural products) with complex chemical structures, yet with simple, sophisticated molecular mechanisms. When natural products exhibit their activities in cells or bodies, they first have to bind or react with a target molecule in/on the cell. The cell membrane is a major target for bioactive compounds. Recently, our understanding of the molecular mechanism of interactions between natural products and membrane lipids progressed with the aid of newly-developed analytical methods. New technology reconnects old compounds with membrane lipids, while new membrane-targeting molecules are being discovered through the screening for antimicrobial potential of natural products. This review article focuses on natural products that bind to eukaryotic membrane lipids, and includes clinically important molecules and key research tools. The chemical diversity of membrane-targeting natural products and the molecular basis of lipid recognition are described. The history of how their mechanism was unveiled, and how these natural products are used in research are also mentioned.
The current trend of increment in the frequency of antifungal resistance has brought research
into an era where new antifungal compounds with novel mechanisms of action are required.
Natural antimicrobial peptides, which are ubiquitous components of innate immunity, represent their
candidature for novel antifungal peptides. Various antifungal peptides have been isolated from different
species ranging from small marine organisms to insects and from various other living species.
Based on these peptides, various mimetics of antifungal peptides have also been synthesized using
non-natural amino acids. Utilization of these antifungal peptides is somehow limited due to their toxic
and unstable nature. This review discusses recent updates and future directions of antifungal peptides,
for taking them to the shelf from the bench.
Bicyclic peptides are conformationally rigid molecules that can bind with high affinity and specificity to their protein target, yet are significantly more protease stable than their linear or monocyclic counterparts. This emerging class of peptides has great potential for the development of novel peptide therapeutics and molecular probes. In this review, we highlight the structural complexity, synthesis/biosynthesis, and biological applications of bridged bicyclic peptides.
Theonellamide A (TNM-A) is an antifungal bicyclic dodecapeptide isolated from a marine sponge Theonella sp. Previous studies have shown that TNM-A preferentially binds to 3β-hydroxysterol-containing membranes and disrupts membrane integrity. In this study, several (1)H NMR-based experiments were performed to investigate the interaction mode of TNM-A with model membranes. First, the aggregation propensities of TNM-A were examined using diffusion ordered spectroscopy; the results indicate that TNM-A tends to form oligomeric aggregates of 2-9 molecules (depending on peptide concentration) in an aqueous environment, and this aggregation potentially influences the membrane-disrupting activity of the peptide. Subsequently, we measured the (1)H NMR spectra of TNM-A with sodium dodecyl sulfate-d25 (SDS-d25) micelles and small dimyristoylphosphatidylcholine (DMPC)-d54/dihexanoylphosphatidylcholine (DHPC)-d22 bicelles in the presence of a paramagnetic quencher Mn(2+). These spectra indicate that TNM-A poorly binds to these membrane mimics without sterol and mostly remains in the aqueous media. In contrast, broader (1)H signals of TNM-A were observed in 10mol% cholesterol-containing bicelles, indicating that the peptide efficiently binds to sterol-containing bilayers. The addition of Mn(2+) to these bicelles also led to a decrease in the relative intensity and further line-broadening of TNM-A signals, indicating that the peptide stays near the surface of the bilayers. A comparison of the relative signal intensities with those of phospholipids showed that TNM-A resides in the lipid-water interface (close to the C2' portion of the phospholipid acyl chain). This shallow penetration of TNM-A to lipid bilayers induces an uneven membrane curvature and eventually disrupts membrane integrity. These results shed light on the atomistic mechanism accounting for the membrane-disrupting activity of TNM-A and the important role of cholesterol in its mechanism of action.
Cholesterol plays important roles in biological membranes. The cellular location where cholesterol molecules work is prerequisite information for understanding their dynamic action. Bioimaging probes for cholesterol molecules would be the most powerful means for unraveling the complex nature of lipid membranes. However, only a limited number of chemical or protein probes have been developed so far for cytological analysis. Here we show that fluorescently-labeled derivatives of theonellamides act as new sterol probes in mammalian cultured cells. The fluorescent probes recognized cholesterol molecules and bound to liposomes in a cholesterol-concentration dependent manner. The probes showed patchy distribution in the plasma membrane, while they stained specific organelle in the cytoplasm. These data suggest that fTNMs will be valuable sterol probes for studies on the role of sterols in the biological membrane under a variety of experimental conditions.
Liposome formation and lipid swelling on platinum electrodes in distilled water and water solutions in d.c. electrical fields have been investigated for different amounts of a negatively charged lipid (mixture from 71% PC, 21.5% PE and 7.5% PS), and a neutral lipid (DMPC). Negatively charged lipids do not form liposomes without field when the thickness of the dried lipid layer is of the order or less than that corresponding to 90 bilayers. The rate and extent of swelling of layers thicker than 90 bilayers is largest on the cathode, smaller without fields and smallest on the anode. The theory, based on the assumption that osmotic and electrostatic forces drive lipid swelling and liposome formation, is in semi-quantitative agreement with the experimental data; in particular, it gives the observed linear dependence of the rate of swelling on the inverse lipid layer thickness. To induce liposome formation for layers thinner than 90 bilayers it was necessary to apply a negative potential which is proportional to the logarithm of the inversed layer thickness. The characteristic critical potential is proportional to RTk/F; R being the gas constant, Tk the absolute temperature and F the Faraday constant. This indicates that redistribution of counterions may be the cause which increases the repulsive electrostatic intermembrane forces to overcome van der Waals attraction. For thicknesses below 10 bilayers, formation of very thin-walled liposomes of narrow size distribution and mean diameter of the order of 30 µm was observed. These liposomes grow in size before detachment and a formula for the kinetics of growth has been derived, which is in very good agreement with the experimental data. The effects of d.c. fields on DMPC swelling are smaller and lead to formation of liposome-like structures of different appearence. Bilayer separation and bending are prerequisites for liposome formation from hydrating lipids. Therefore, a possible molecular mechanism is that membranes should be destabilized to bend and fuse to form liposomes. This requires the right proportion between structured regions, in the form of bilayers, and defects and/or non-bilayer structures, and in many cases external constraints, in particular, electrical fields.
Fluorescence spectroscopy-based techniques using conventional fluorimeters have been extensively applied since the late 1960s
to study different aspects of membrane-related phenomena, i.e., mainly relating to lipid-lipid and lipid-protein (peptide)
interactions. Even though fluorescence spectroscopy approaches provide very valuable structurally and dynamically related
information on membranes, they generally produce mean parameters from data collected on bulk solutions of many vesicles and
lack direct information on the spatial organization at the level of single membranes, a quality that can be provided by microscopy-related
techniques. In this chapter, I will attempt to summarize representative examples concerning how microscopy (which provides
information on membrane lateral organization by direct visualization) and spectroscopy techniques (which provides information
about molecular interaction, order and microenvironment) can be combined to give a powerful new approach to study membrane-related
phenomena. Additionally along this chapter, it will be discussed how membrane model systems can be further utilized to gain
information about particular membrane-related process like protein(peptide)/membrane interactions.
KeywordsGiant unilamellar vesicles-Membrane lateral structure-Membrane-peptide interaction-Polarity sensitive probes-Fluorescence microscopy
Shallow hydrophobic insertions and crescent-shaped BAR scaffolds promote membrane curvature. Here, we investigate membrane fission by shallow hydrophobic insertions quantitatively and mechanistically. We provide evidence that membrane insertion of the ENTH domain of epsin leads to liposome vesiculation, and that epsin is required for clathrin-coated vesicle budding in cells. We also show that BAR-domain scaffolds from endophilin, amphiphysin, GRAF, and β2-centaurin limit membrane fission driven by hydrophobic insertions. A quantitative assay for vesiculation reveals an antagonistic relationship between amphipathic helices and scaffolds of N-BAR domains in fission. The extent of vesiculation by these proteins and vesicle size depend on the number and length of amphipathic helices per BAR domain, in accord with theoretical considerations. This fission mechanism gives a new framework for understanding membrane scission in the absence of mechanoenzymes such as dynamin and suggests how Arf and Sar proteins work in vesicle scission.
The chemical investigation of an Indonesian specimen of Theonella swinhoei afforded four aurantosides, one of which, aurantoside J (5), is a new compound. The structure of this metabolite, exhibiting the unprecedented N-α-glycosidic linkage between the pentose and the tetramate units, has been determined through detailed spectroscopic analysis. The four obtained aurantosides have been tested against five fungal strains (four Candida and one Fusarium) responsible of invasive infections in immuno-compromised patients. The non-cytotoxic aurantoside I (4) was the single compound to show an excellent potency against all the tested strains, thus providing valuable insights about the antifungal potential of this class of compounds and the structure-activity relationships.
Lithistid sponges are known to produce a diverse array of compounds ranging from polyketides, cyclic and linear peptides, alkaloids, pigments, lipids, and sterols. A majority of these structurally complex compounds have very potent and interesting biological activities. It has been a decade since a thorough review has been published that summarizes the literature on the natural products reported from this amazing sponge order. This review provides an update on the current taxonomic classification of the Lithistida, describes structures and biological activities of 131 new natural products, and discusses highlights from the total syntheses of 16 compounds from marine sponges of the Order Lithistida providing a compilation of the literature since the last review published in 2002.
In our ongoing search for new pharmacologically active leads from Solomon organisms, we have examined the sponge Theonella swinhoei. Herein we report the isolation and structure elucidation of swinholide A (1) and one new macrolide, swinholide J (2). Swinholide J is an unprecedented asymmetric 44-membered dilactone with an epoxide functionality in half of the molecule. The structural determination was based on extensive interpretation of high-field NMR spectra and HRESIMS data. Swinholide J displayed potent in vitro cytotoxicity against KB cells (human nasopharynx cancer) with an IC(50) value of 6 nM.
Linking bioactive compounds to their cellular targets is a central challenge in chemical biology. Here we report the mode of action of theonellamides, bicyclic peptides derived from marine sponges. We generated a chemical-genomic profile of theonellamide F using a collection of fission yeast strains in which each open reading frame (ORF) is expressed under the control of an inducible promoter. Clustering analysis of the Gene Ontology (GO) terms associated with the genes that alter drug sensitivity suggested a mechanistic link between theonellamide and 1,3-beta-D-glucan synthesis. Indeed, theonellamide F induced overproduction of 1,3-beta-D-glucan in a Rho1-dependent manner. Subcellular localization and in vitro binding assays using a fluorescent theonellamide derivative revealed that theonellamides specifically bind to 3beta-hydroxysterols, including ergosterol, and cause membrane damage. The biological activity of theonellamides was alleviated in mutants defective in ergosterol biosynthesis. Theonellamides thus represent a new class of sterol-binding molecules that induce membrane damage and activate Rho1-mediated 1,3-beta-D-glucan synthesis.
We present a yeast chemical-genomics approach designed to identify genes that when mutated confer drug resistance, thereby providing insight about the modes of action of compounds. We developed a molecular barcoded yeast open reading frame (MoBY-ORF) library in which each gene, controlled by its native promoter and terminator, is cloned into a centromere-based vector along with two unique oligonucleotide barcodes. The MoBY-ORF resource has numerous genetic and chemical-genetic applications, but here we focus on cloning wild-type versions of mutant drug-resistance genes using a complementation strategy and on simultaneously assaying the fitness of all transformants with barcode microarrays. The complementation cloning was validated by mutation detection using whole-genome yeast tiling microarrays, which identified unique polymorphisms associated with a drug-resistant mutant. We used the MoBY-ORF library to identify the genetic basis of several drug-resistant mutants and in this analysis discovered a new class of sterol-binding compounds.
Model systems such as black lipid membranes or conventional uni- or multilamellar liposomes are commonly used to study membrane properties and structure. However, the construction and dimensions of these models excluded their direct optical microscopic observation. Since the introduction of the simple method of liposome electroformation in alternating electric field giant unilamellar vesicles (GUVs) have become an important model imitating biological membranes. Due to the average diameter of GUVs reaching up to 100 microm, they can be easily observed under a fluorescent or confocal microscope provided that the appropriate fluorescent probe was incorporated into the lipid phase during vesicle formation. GUVs can be formed from different lipid mixtures and they are stable in a wide range of physical conditions such as pH, pressure or temperature. This mini-review presents information about the methods of GUV production and their usage. Particularly, the use of GUVs in studying lipid phase separation and the appearance and behavior of lipid domains (rafts) in membranes is discussed but also other examples of GUVs use in membrane research are given. The experience of the authors in setting up the GUV-forming equipment and production of GUVs is also presented.
Two unprecedented cyclic peptides, solomonamides A and B, were Isolated from the marine sponge Theonella swinhoel. The structures were elucidated on the basis of comprehensive 1D and 2D NMR analysis and high-resolution mass spectrometry. A combined approach, involving Marfey's method, QM J based analysis, and DFT J/C-13 calculations, was used for establishing the absolute configuration of the entire molecule. Solomonamide A showed in vivo anti-inflammatory activity.
Roles of lipids in the cell membrane are poorly understood. This is partially due to the lack of methodologies, for example, tool chemicals that bind to specific membrane lipids and modulate membrane function. Theonellamides (TNMs), marine sponge-derived peptides, recognize 3β-hydroxysterols in lipid membranes and induce major morphological changes in cultured mammalian cells through as yet unknown mechanisms. Here, we show that TNMs recognize cholesterol-containing liquid-disordered domains and induce phase separation in model lipid membranes. Modulation of membrane order was also observed in living cells following treatment with TNM-A, in which cells shrank considerably in a cholesterol-, cytoskeleton-, and energy-dependent manner. These findings present a previously unrecognized mode of action of membrane-targeting natural products. Meanwhile, we demonstrated the importance of membrane order, which is maintained by cholesterol, for proper cell morphogenesis.
Copyright © 2015 Elsevier Ltd. All rights reserved.
Short cationic, amphipathic antimicrobial peptides are multi-functional molecules that have roles in host defense as direct microbicides and modulators of the immune response. While a general mechanism of microbicidal activity involves the selective disruption and permeabilization of cell membranes, the relationships between peptide sequence and membrane activity are still under investigation. Here, we review the diverse functions that AMPs collectively have in host defense, and show that these functions can be multiplexed with a membrane mechanism of activity derived from the generation of negative Gaussian membrane curvature. As AMPs preferentially generate this curvature in model bacterial cell membranes, the selective generation of negative Gaussian curvature provides AMPs with a broad mechanism to target microbial membranes. The amino acid constraints placed on AMPs by the geometric requirement to induce negative Gaussian curvature are consistent with known AMP sequences. This ‘saddle-splay curvature selection rule’ is not strongly restrictive so AMPs have significant compositional freedom to multiplex membrane activity with other useful functions. The observation that certain proteins involved in cellular processes which require negative Gaussian curvature contain domains with similar motifs as AMPs, suggests this rule may be applicable to other curvature-generating proteins. Since our saddle-splay curvature design rule is based upon both a mechanism of activity and the existing motifs of natural AMPs, we believe it will assist the development of synthetic antimicrobials.
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Biological membrane fission requires protein-driven stress. The guanosine triphosphatase (GTPase) dynamin builds up membrane
stress by polymerizing into a helical collar that constricts the neck of budding vesicles. How this curvature stress mediates
nonleaky membrane remodeling is actively debated. Using lipid nanotubes as substrates to directly measure geometric intermediates
of the fission pathway, we found that GTP hydrolysis limits dynamin polymerization into short, metastable collars that are
optimal for fission. Collars as short as two rungs translated radial constriction to reversible hemifission via membrane wedging
of the pleckstrin homology domains (PHDs) of dynamin. Modeling revealed that tilting of the PHDs to conform with membrane
deformations creates the low-energy pathway for hemifission. This local coordination of dynamin and lipids suggests how membranes
can be remodeled in cells.
Theonellamides (TNMs) are members of a distinctive family of antifungal and cytotoxic bicyclic dodecapeptides isolated from the marine sponge Theonella sp. Recently, it has been shown that TNMs recognize 3β-hydroxysterol-containing membranes, induce glucan overproduction, and damage cellular membranes. However, to date, the detailed mode of sterol-binding at a molecular level has not been determined. In this study, to gain insight into the mechanism of sterol-recognition of TNM in lipid bilayers, surface plasmon resonance (SPR) experiments and solid state deuterium nuclear magnetic resonance (2H NMR) measurements were performed on theonellamide A (TNM-A). SPR results revealed that the incorporation of 10 mol% of cholesterol or ergosterol into 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) membranes significantly enhances the affinity of the peptide for the membrane, particularly in the initial binding process, to the membrane surface. These findings, together with the fact that binding of TNM-A to epicholesterol (3α-cholesterol)-containing and to pure POPC liposomes was comparably low, confirmed the preference of the peptide toward the 3β-hydroxysterol-containing membranes. To further establish the complex formation of TNM-A with 3β-hydroxysterols in lipid bilayers, solid-state 2H NMR measurements were carried out using deuterium-labeled cholesterol, ergosterol, or epicholesterol. The 2H NMR spectra showed that TNM-A significantly inhibits the fast rotational motion of cholesterol and ergosterol, but not epicholesterol, therefore verifying the direct complexation between TNM-A and 3β-hydroxysterols in lipid bilayers. This study demonstrates that TNM-A directly recognizes the 3β-OH moiety of sterols, which greatly facilitates its binding to bilayer membranes.
Theopalauamide (1) is the major bicyclic peptide from the symbiotic filamentous eubacteria that are found in the interior of Theonella swinhoei from Palau. It was also isolated from T. swinhoei from Mozambique. The structure of theopalauamide was determined by analysis of spectroscopic data, and its stereochemistry was determined by chemical degradation and analysis of the products by chiral GC-MS. The minor peptide, isotheopalauamide (2), was shown to be a stable conformational isomer that was formed by TFA-catalyzed equilibration during the isolation procedure.
Five new cytotoxic peptides, related to theonellamide F (1), were isolated from a marine sponge Theonella sp. Theonellamide A (2) and B (3) differ from F (1) in three amino acid residues. Additionally, theonellamide A (2) bears a beta-D-galactose linked to the free imidazole nitrogen; Theonellamide C (4) is debromo 1. Theonellamide D (5) and E (6) are the beta-L-arabinoside and beta-D-galactoside of 1. Structures 2-6 were assigned on the basis of spectral data and chromatographic analyses of degradation products.
Theonegramide (2) is an antifungal glycopeptide consisting of arabinose joined to a bicyclic dodecapeptide that contains the unusual amino acids β-hydroxyasparagine, isoserine, α-aminoadipic acid, 4′-bromo-3-methylphenylalanine, 3-amino-4-hydroxy-6-methyl-8-phenylocta-5,7-dienoic acid, and the same histidinoalanine moiety previously observed in theonellamide F (1). The structure of theonegramide (2) was elucidated by analysis of spectral data and its absolute configuration was determined by GC-MS analysis using a chiral column packing.
A novel antifungal peptide, theonellamide F, was isolated from a marine sponge, genus Theonella. It is a dodecapeptide composed of L-Asn, L-aThr, two residues of L-Ser, L-Phe, βAla, (2S,3R)-3-hydroxyasparagine, (2S,4R)-2-amino-4-hydroxyadipic acid, τ-L-histidino-D-alanine, L-p-bromophenylalanine, and (3S,4S,5E,7E)-3-amino-4-hydroxy-6-methyl-8-(p-bromophenyl)-5,7-octadienoic acid. Its bicyclic structure including absolute stereochemistry was unequivocally determined as 1, which contains an unprecedented histidinoalanine bridge.
NMR is a versatile tool for studying interactions between antimicrobial peptides and lipid membranes. Different approaches using both liquid state and solid state NMR are outlined here, with an emphasis on solid state NMR methods, to study the structures of antimicrobial peptides in lipid bilayers as well as the effect of these peptides on model membranes. Different NMR techniques for observing both peptides and lipids are explained, including 2H, 13C, 15N, and 19F labels, or natural abundance 1H, 13C, or 31P. Previous studies in the field are extensively reviewed in easily accessible tables. © 2004 Wiley Periodicals, Inc. Concepts Magn Reson 23A: 89–120, 2004.
Solid-state NMR spectroscopic techniques provide valuable information about the structure, dynamics and topology of membrane-inserted polypeptides. In particular antimicrobial peptides (or 'host defence peptides') have early on been investigated by solid-state NMR spectroscopy and many technical innovations in this domain have been developed with the help of these compounds when reconstituted into oriented phospholipid bilayers. Using solid-state NMR spectroscopy it could be shown for the first time that magainins or derivatives thereof exhibit potent antimicrobial activities when their cationic amphipathic helix is oriented parallel to the bilayer surface, a configuration found in later years for many other linear cationic amphipathic peptides. In contrast transmembrane alignments or lipid-dependent tilt angles have been found for more hydrophobic sequences such as alamethicin or β-hairpin antimicrobials. This review presents various solid-state NMR approaches and develops the basic underlying concept how angular information can be obtained from oriented samples. It is demonstrated how this information is used to calculate structures and topologies of peptides in their native liquid-disordered phospholipid bilayer environment. Special emphasis is given to discuss which NMR parameters provide the most complementary information, the minimal number of restraints needed and the effect of motions on the analysis of the NMR spectra. Furthermore, recent (31)P and (2)H solid-state NMR measurements of lipids are presented including some unpublished data which aim at investigating the morphological and structural changes of oriented or non-oriented phospholipids. Finally the structural models that have been proposed for the mechanisms of action of these peptides will be presented and discussed in view of the solid-state NMR and other biophysical experiments.
Two unprecedented cyclic peptides, solomonamides A and B, were isolated from the marine sponge Theonella swinhoei. The structures were elucidated on the basis of comprehensive 1D and 2D NMR analysis and high-resolution mass spectrometry. A combined approach, involving Marfey's method, QM J based analysis, and DFT J/(13)C calculations, was used for establishing the absolute configuration of the entire molecule. Solomonamide A showed in vivo anti-inflammatory activity.
The finding of new PXR modulators as potential leads for treatment of human disorders characterized by dysregulation of innate immunity and with inflammation is of wide interest. In this paper, we report the identification of the first example of natural marine PXR agonists, solomonsterols A and B, from a Theonella swinhoei sponge. The structures were determined by interpretation of NMR and ESIMS data, and the putative binding mode to PXR has been obtained through docking calculations.
Antimicrobial peptides (AMPs) have been studied for three decades, and yet a molecular understanding of their mechanism of action is still lacking. Here we summarize current knowledge for both synthetic vesicle experiments and microbe experiments, with a focus on comparisons between the two. Microbial experiments are done at peptide to lipid ratios that are at least 4 orders of magnitude higher than vesicle-based experiments. To close the gap between the two concentration regimes, we propose an "interfacial activity model", which is based on an experimentally testable molecular image of AMP-membrane interactions. The interfacial activity model may be useful in driving engineering and design of novel AMPs.
A new sulfated cyclic depsipeptide, termed mutremdamide A, and six new highly N-methylated peptides, termed koshikamides C-H, were isolated from different deep-water specimens of Theonella swinhoei and Theonella cupola. Their structures were determined using extensive 2D NMR, ESI, or CDESI and QTOF-MS/MS experiments and absolute configurations established by quantum mechanical calculations, advanced Marfey's method, and chiral HPLC. Mutremdamide A displays a rare 2-amino-3-(2-hydroxyphenyl)propanoic acid and a new N(delta)-carbamoyl-beta-sulfated asparagine. Koshikamides C-E are linear undecapeptides, and koshikamides F-H are 17-residue depsipeptides containing a 10-residue macrolactone. Koshikamides F and G differ from B and H in part by the presence of the conjugated unit 2-(3-amino-5-oxopyrrolidin-2-ylidene)propanoic acid. Cyclic koshikamides F and H inhibited HIV-1 entry at low micromolar concentrations while their linear counterparts were inactive. The Theonella collections studied here are distinguished by co-occurrence of mutremdamide A, koshikamides, and theonellamides, the combination of which appears to define a new Theonella chemotype that can be found in deeper waters.
Numerous data have been collected on lipid-binding amphipathic helices involved in membrane-remodeling machineries and vesicular transport. Here we describe how, with regard to lipid composition, the physicochemical features of some amphipathic helices explain their ability to recognize membrane curvature or to participate in membrane remodeling. We propose that sensing highly-curved membranes requires that the polar and hydrophobic faces of the helix do not cooperate in lipid binding. A more detailed description of the interaction between amphipathic helices and lipids is however needed; notably to explain how new helices contribute to detection of modest changes in curvature or even negative curvature.
Abstract: Bioassay-guided isolation from the ethanol extract of a marine sponge Theonella sp. collected in Palau yielded bistheonellide A, which strongly inhibited the colony formation of Chinese hamster V79 cells (EC50 = 6.8 nM). Bistheonellide A is an actinpolymerization inhibitor and was suggested to control cytokine production. Therefore, we attempted to detect an effect of this compound on IL-8 production in PMA-stimulated HL- 60 cells. Interestingly, bistheonellide A did not modulate the production of IL-8 under cytotoxic concentrations as determined by LDH analysis. Although the correlation between the inhibition of microtubule assembly and the stimulation of IL-8 production has been observed for several compounds, the polymerization of actin was not related to an IL-8 production in the case of bistheonellide A. It will be suggested that the actin polymerization is not involved in the IL-8 production system.
Polytheonamides A and B are highly cytotoxic polypeptides with 48 amino acid residues isolated from the marine sponge, Theonella swinhoei. The structure of polytheonamide B was determined by spectral and chemical methods, especially extensive 2D NMR experiments, which resulted in the unprecedented polypeptide structure; the N-terminal glycine blocked with a 5,5-dimethyl-2-oxo-hexanoyl group, the presence of eight tert-leucine, three beta-hydroxyvaline, six gamma-N-methylasparagine, two gamma-N-methyl-beta-hydroxyasparagine, and beta,beta-dimethymethionine sulfoxide residues. More significantly, it has the sequence of alternating D- and L-amino acids. Polytheonamide A is an epimer of polytheonamide B differing only in the stereochemistry of the sulfoxide of the 44(th) residue.
Theopapuamide (1), a new cytotoxic peptide, has been isolated from the lithistid sponge Theonella swinhoei from Papua New Guinea. The structure was established by analysis of NMR, mass spectrometry, and chemical methods. The undecapeptide (1) contains several unusual amino acid residues, of which the occurrence of beta-methoxyasparagine and 4-amino-5-methyl-2,3,5-trihydroxyhexanoic acid (Amtha) is unprecedented in natural peptides. Compound 1 also contains an amide-linked fatty acid moiety, 3-hydroxy-2,4,6-trimethyloctanoic acid (Htoa). Theopapuamide (1) was cytotoxic against CEM-TART and HCT-116 cell lines, with EC50 values of 0.5 and 0.9 microM, respectively.
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