Recent Results in Alamethicin Research

Kredics, László; Szekeres, András; Czifra, Dorina; Vágvölgyi, Csaba; Leitgeb, Balázs

doi:10.1002/cbdv.201200390

Cited by 29 publications

(26 citation statements)

References 129 publications

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“…In comparison, acute administration of 1 blocked calcium uptake by the mitochondria in a concentration-dependent manner (0.05-0.1 μM). Although the indiscriminate membrane-disrupting agent alamethicin also had this effect on purified mitochondria (55) (Fig. 7I), our previous observations that the compound does not enter cells at up to 10 μM meant that this is not a relevant cytotoxic mechanism for this classical peptaibol at these concentrations.…”

Section: Partial Hydrolysis Of Gichigamins Reveals Structural Requirementioning

confidence: 74%

Unique amalgamation of primary and secondary structural elements transform peptaibols into potent bioactive cell-penetrating peptides

Risinger²,

Mitchell

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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Mass-spectrometry-based metabolomics and molecular phylogeny data were used to identify a metabolically prolific strain of Tolypocladium that was obtained from a deep-water Great Lakes sediment sample. An investigation of the isolate's secondary metabolome resulted in the purification of a 22-mer peptaibol, gichigamin A (1). This peptidic natural product exhibited an amino acid sequence including several β-alanines that occurred in a repeating ααβ motif, causing the compound to adopt a unique right-handed 3 11 helical structure. The unusual secondary structure of 1 was confirmed by spectroscopic approaches including solution NMR, electronic circular dichroism (ECD), and single-crystal X-ray diffraction analyses. Artificial and cell-based membrane permeability assays provided evidence that the unusual combination of structural features in gichigamins conferred on them an ability to penetrate the outer membranes of mammalian cells. Compound 1 exhibited potent in vitro cytotoxicity (GI 50 0.55 ± 0.04 μM) and in vivo antitumor effects in a MIA PaCa-2 xenograft mouse model. While the primary mechanism of cytotoxicity for 1 was consistent with ion leakage, we found that it was also able to directly depolarize mitochondria. Semisynthetic modification of 1 provided several analogs, including a C-terminus-linked coumarin derivative (22) that exhibited appreciably increased potency (GI 50 5.4 ± 0.1 nM), but lacked ion leakage capabilities associated with a majority of naturally occurring peptaibols such as alamethicin. Compound 22 was found to enter intact cells and induced cell death in a process that was preceded by mitochondrial depolarization.peptaibol | fungi | natural products | gichigamin | mitochondria N atural product chemical research has evolved in many extraordinary ways over the last decade largely due to the integration of powerful and accessible analytical chemistry (1, 2) and molecular biology (3, 4) tools. Some of these techniques have been applied toward the creation of natural product big-data sets in which genomic, metagenomic, metabolomic, and proteomic approaches are combined to obtain new insights into nature's remarkable chemical bounty (4-8). The predictive capabilities of these methods have helped guide efforts in several fertile areas of natural products research including the DNA cross-linking enediynes (9), bioactive cyanobacterial metabolites (10, 11), cytotoxic pentangular polyphenols (12), and thiopeptide antibiotics (13).The integration of analytical and molecular approaches is an effective way to find new natural product analogs that exhibit enhanced biological activities and improved pharmacological attributes (e.g., greater potency, better solubility, enhanced therapeutic index, etc.), but it also has the potential to uncover compounds with activities that are markedly dissimilar to other members of the queried chemical class. Nature is well known for its chemical repurposing capabilities, which allow populations of organisms to evolve new biological functions from existing molecular sc...

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Section: Partial Hydrolysis Of Gichigamins Reveals Structural Requirementioning

confidence: 74%

Unique amalgamation of primary and secondary structural elements transform peptaibols into potent bioactive cell-penetrating peptides

Risinger²,

Mitchell

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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“…Alamethicins (ALMs) belong to a family of antibiotic peptides (peptaibols) of fungal origin that are known to selfassociate upon binding to lipid membranes (reviewed in (1)(2)(3)(4)(5)(6)(7)(8)). The most extensively studied isoform, ALM F50/5, is characterized by a linear sequence of 19 amino acids, eight of which are a-aminoisobutyric acid residues.…”

Section: Introductionmentioning

confidence: 99%

Alamethicin Supramolecular Organization in Lipid Membranes from 19F Solid-State NMR

Salnikov

Raya

Zotti

et al. 2016

Biophysical Journal

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Alamethicins (ALMs) are antimicrobial peptides of fungal origin. Their sequences are rich in hydrophobic amino acids and strongly interact with lipid membranes, where they cause a well-defined increase in conductivity. Therefore, the peptides are thought to form transmembrane helical bundles in which the more hydrophilic residues line a water-filled pore. Whereas the peptide has been well characterized in terms of secondary structure, membrane topology, and interactions, much fewer data are available regarding the quaternary arrangement of the helices within lipid bilayers. A new, to our knowledge, fluorine-labeled ALM derivative was prepared and characterized when reconstituted into phospholipid bilayers. As a part of these studies, C 19 F 3 -labeled compounds were characterized and calibrated for the first time, to our knowledge, for 19 F solid-state NMR distance and oligomerization measurements by centerband-only detection of exchange (CODEX) experiments, which opens up a large range of potential labeling schemes. The 19 F-19 F CODEX solid-state NMR experiments performed with ALM in POPC lipid bilayers and at peptide/lipid ratios of 1:13 are in excellent agreement with molecular-dynamics calculations of dynamic pentameric assemblies. When the peptide/lipid ratio was lowered to 1:30, ALM was found in the dimeric form, indicating that the supramolecular organization is tuned by equilibria that can be shifted by changes in environmental conditions.

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“…It was discovered in the late 1960’s and has been studied as a model pore-forming peptide for decades [1, 2]. Alamethicin has an unusual primary sequence that includes non-standard amino acids and C- and N-terminal modifications.…”

Section: Introductionmentioning

confidence: 99%

Single-cell, time-resolved study of the effects of the antimicrobial peptide alamethicin on Bacillus subtilis

Barns

Weisshaar

2016

Biochimica et Biophysica Acta (BBA) - Biomembranes

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Alamethicin is a well studied antimicrobial peptide (AMP) that kills Gram positive bacteria. It forms narrow, barrel stave pores in planar lipid bilayers. We present a detailed, time-resolved microscopy study of the sequence of events during the attack of alamethicin on individual, live Bacillus subtilis cells expressing GFP in the cytoplasm. At the minimum inhibitory concentration (MIC), the first observed symptom is the halting of growth, as judged by the plateau in measured cell length vs time. The data strongly suggest that this growth-halting event occurs prior to membrane permeabilization. Gradual degradation of the proton motive force, inferred from a decrease in pH-dependent GFP fluorescence intensity, evidently begins minutes later and continues over about 5 min. There follows abrupt permeabilization of the cytoplasmic membrane to the DNA stain Sytox Orange and concomitant loss of small osmolytes, causing observable cell shrinkage presumably due to decreased turgor pressure. This permeabilization of the cytoplasmic membrane occurs uniformly across the entire membrane, not locally, on a timescale of 5 s or less. GFP gradually leaks out of the cell envelope, evidently impeded by the shrunken peptidoglycan layer. Disruption of the cell envelope by alamethicin occurs in stages, with larger and larger species permeating the envelope as time evolves over tens of minutes. Some of the observed symptoms are consistent with formation of barrel stave pores, but the data do not rule out “chaotic pore” or “carpet” mechanisms. We contrast the effects of alamethicin and the human cathelicidin LL-37 on B. subtilis.

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Recent Results in Alamethicin Research

Cited by 29 publications

References 129 publications

Unique amalgamation of primary and secondary structural elements transform peptaibols into potent bioactive cell-penetrating peptides

Unique amalgamation of primary and secondary structural elements transform peptaibols into potent bioactive cell-penetrating peptides

Alamethicin Supramolecular Organization in Lipid Membranes from 19F Solid-State NMR

Single-cell, time-resolved study of the effects of the antimicrobial peptide alamethicin on Bacillus subtilis

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