Soil sustains human life by nourishing crops, storing food sources, and housing microbes, which may affect the nutrition and biosynthesis of secondary metabolites, some of which are used as drugs. To identify lead compounds for a new class of drugs, we collected soil-derived fungal strains from various environments, including urban areas. As various human pathogens are assumed to influence the biosynthetic pathways of metabolites in soil fungi, leading to the production of novel scaffolds, we focused our work on densely populated urban areas and tourist attractions. A soil-derived fungal extract library was screened against MDA-MB-231 cells to derive their cytotoxic activity. Notably, 10 μg/mL of the extract of Trichoderma guizhouense (DS9-1) was found to exhibit an inhibitory effect of 71%. Fractionation, isolation, and structure elucidation efforts led to the identification of nine new peptaibols, trichoguizaibols A-I (1-9), comprising 14 amino acid residues (14-AA peptaibols), and three new peptaibols, trichoguizaibols J-L (10-12), comprising 18 amino acid residues (18-AA peptaibols). The chemical structures of 1-12 were determined based on their 1D and 2D NMR spectra, HRESIMS, electronic circular dichroism data, and results of the advanced Marfey\'s method. The 18-AA peptaibols were found to exhibit cytotoxicity against MDA-MB-231, SK-Hep1, SKOV3, DU145, and HCT116 cells greater than that of the 14-AA peptaibols. Among these compounds, 10-12 exhibited potent sub-micromolar IC50 values. These results are expected to shed light on a new direction for developing novel scaffolds as anticancer agents.

Five new lipopeptaibols (1-5) and eight new 19-residue peptaibols (8-15) along with two known lipopeptaibols, lipovelutibols C (6) and D (7) were isolated from Trichoderma strigosum. The planar structures of the newly discovered peptaibols (1-5, 8-15) were elucidated using 1D and 2D NMR, and UPLC-MS/MS data. The absolute configurations for new peptaibols (1-5, 8-15) were elucidated using the advanced Marfey\'s method and GITC (2,3,4,6-tetra-O-acetyl-β-d-glucopyranosyl isothiocyanate) derivatization. Through analysis of CD spectra, these peptabols were found to have right-handed helical conformations. While most of the new compounds were significantly more active than the positive control, 9, 10, 12, and 15 containing Ser and Leu at positions 10 and 11, respectively, were the most cytotoxic against MDA-MB-231, SNU449, SKOV3, DU145, and HCT116 cancer cell lines, and the 19-residue peptaibols were generally more potent than lipopeptaibols.

Peptaibols are a class of short peptides, typically 7 to 20 amino acids long, characterized by noncanonical amino acid residues such as aminoisobutyric acid (Aib). Although the helix length is shorter than the membrane thickness, the 11-residue peptaibol trichorovin-XII (TV-XII) can form ion channels in membranes. Assuming that a higher proportion of isoleucine (Ile) relative to leucine (Leu) residues is crucial for maintaining the ion channel activity of TV-XII, peptide analogs of TV-XII with varying Ile content were designed, synthesized, and evaluated. The secondary structure of all derivatives under hydrophobic conditions was confirmed by CD measurement as an α-helix-like β-bend ribbon spiral structure. The most stable ion channel activity was found in compound 4a with maximum Ile. Furthermore, the C-terminal Ile analog showed greater ion channel activity compared to the Leu analog. This suggests that the choice between Leu and Ile can influence the expression of ion channel activity, which will be crucial for the de novo designed functional peptides.

Plant diseases caused by Pseudomonas syringae are essentially controlled in the field with the use of copper-based products and antibiotics, raising environmental and safety concerns. Antimicrobial peptides (AMPs) derived from fungi may represent a sustainable alternative to those chemicals. Trichogin GA IV, a non-ribosomal, 11-residue long AMP naturally produced by the fungus Trichoderma longibrachiatum has the ability to insert into phospholipidic membranes and form water-filled pores, thereby perturbing membrane integrity and permeability. In previous studies, peptide analogs modified at the level of specific residues were designed to be water-soluble and active against plant pathogens. Here, we studied the role of glycine-to-lysine substitutions and of the presence of a C-terminal leucine amide on bioactivity against Pseudomonas syringae bacteria. P. syringae diseases affect a wide range of crops worldwide, including tomato and kiwifruit. Our results show that trichogin GA IV analogs containing two or three Gly-to-Lys substitutions are highly effective in vitro against P. syringae pv. tomato (Pst), displaying minimal inhibitory and minimal bactericidal concentrations in the low micromolar range. The same analogs are also able to inhibit in vitro the kiwifruit pathogen P. syringae pv. actinidiae (Psa) biovar 3. When sprayed on tomato plants 24 h before Pst inoculation, only tri-lysine containing analogs were able to significantly reduce bacterial titers and symptom development in infected plants. Our results point to a positive correlation between the number of lysine substitutions and the antibacterial activity. This correlation was supported by microscopy analyses performed with mono-, di- and tri-Lys containing analogs that showed a different degree of interaction with Pst cells and ultrastructural changes that culminated in cell lysis.

Bergofungin D is a helical peptide of the peptaibol family consisting of 14 amino acids, six of which are the helix inducer aminoisobutyric acid (Aib). In the second third of the sequence, a hydroxyproline causes a bending of the helix and a disruption of the hydrogen bond network, and Aib7 is the only amino acid in this region involved in the hydrogen bond network. Therefore, modification of this residue can serve as a probe to monitor the effect of introducing amino acid substitutions on this more fragile helical turn. To validate this approach, we simplified the original bergofungin D by reducing the number of non-classical amino acids, replacing the (R)-isovaleric acid by its enantiomer or an Aib and the hydroxyproline with a proline, respectively, without affecting its secondary structure. Within the modified structure, we replaced Aib7-Aib8 by its 1,2,3-triazolodipeptide equivalent or Aib7 by a serine or a dehydrobutyrine. We have reported and analyzed five crystal structures, three of which are new, demonstrating the usefulness of the modified bergofungin D as a probe for monitoring the introduction of amino acid substitutions within a helical structure.

Perturbations in bilayer material properties (thickness, lipid intrinsic curvature and elastic moduli) modulate the free energy difference between different membrane protein conformations, thereby leading to changes in the conformational preferences of bilayer-spanning proteins. To further explore the relative importance of curvature and elasticity in determining the changes in bilayer properties that underlie the modulation of channel function, we investigated how the micelle-forming amphiphiles Triton X-100, reduced Triton X-100 and the HII lipid phase promoter capsaicin modulate the function of alamethicin and gramicidin channels. Whether the amphiphile-induced changes in intrinsic curvature were negative or positive, amphiphile addition increased gramicidin channel appearance rates and lifetimes and stabilized the higher conductance states in alamethicin channels. When the intrinsic curvature was modulated by altering phospholipid head group interactions, however, maneuvers that promote a negative-going curvature stabilized the higher conductance states in alamethicin channels but destabilized gramicidin channels. Using gramicidin channels of different lengths to probe for changes in bilayer elasticity, we found that amphiphile adsorption increases bilayer elasticity, whereas altering head group interactions does not. We draw the following conclusions: first, confirming previous studies, both alamethicin and gramicidin channels are modulated by changes in lipid bilayer material properties, the changes occurring in parallel yet differing dependent on the property that is being changed; second, isolated, negative-going changes in curvature stabilize the higher current levels in alamethicin channels and destabilize gramicidin channels; third, increases in bilayer elasticity stabilize the higher current levels in alamethicin channels and stabilize gramicidin channels; and fourth, the energetic consequences of changes in elasticity tend to dominate over changes in curvature.

Eco-friendly bioherbicides are urgently needed for managing the problematic weed Amaranthus retroflexus. A mass spectrometry- and bioassay-guided screening approach was employed to identify phytotoxic secondary metabolites from fungi for the development of such bioherbicides. This effort led to the discovery of six phytotoxic 16-residue peptaibols, including five new compounds (2-6) and a known congener (1), from Emericellopsis sp. XJ1056. Their planar structures were elucidated through the analysis of tandem mass and NMR spectroscopic data. The absolute configurations of the chiral amino acids were determined by advanced Marfey\'s method and chiral-phase liquid chromatography-mass spectrometry (LC-MS) analysis. Bioinformatic analysis and targeted gene disruption identified the biosynthetic gene cluster for these peptaibols. Compounds 1 and 2 significantly inhibited the radicle growth of A. retroflexus seedlings, and 1 demonstrated potent postemergence herbicidal activity against A. retroflexus while exhibiting minimal toxicity to Sorghum bicolor. Structure-activity relationship analysis underscored the importance of trans-4-hydroxy-l-prolines at both the 10th and 13th positions for the herbicidal activities of these peptaibols.

From the fungus Trichoderma sp., we isolated seven novel 18-residue peptaibols, neoatroviridins E-K (1-7), and six new 14-residue peptaibols, harzianins NPDG J-O (8-13). Additionally, four previously characterized 18-residue peptaibols neoatroviridins A-D (14-17) were also identified. The structural configurations of the newly identified peptaibols (1-13) were determined by comprehensive nuclear magnetic resonance (NMR) and high-resolution electrospray ionization tandem mass spectrometry (HR-ESI-MS/MS) data. Their absolute configurations were further determined using Marfey\'s method. Notably, compounds 12 and 13 represent the first 14-residue peptaibols containing an acidic amino acid residue. In antimicrobial assessments, all 18-residue peptaibols (1-7, 14-17) exhibited moderate inhibitory activities against Staphylococcus aureus 209P, with minimum inhibitory concentration (MIC) values ranging from 8-32 μg·mL-1. Moreover, compound 9 exhibited moderate inhibitory effect on Candida albicans FIM709, with a MIC value of 16 μg·mL-1.

Our previous study identified 52 antiplasmodial peptaibols isolated from fungi. To understand their antiplasmodial mechanism of action, we conducted phenotypic assays, assessed the in vitro evolution of resistance, and performed a transcriptome analysis of the most potent peptaibol, HZ NPDG-I. HZ NPDG-I and 2 additional peptaibols were compared for their killing action and stage dependency, each showing a loss of digestive vacuole (DV) content via ultrastructural analysis. HZ NPDG-I demonstrated a stepwise increase in DV pH, impaired DV membrane permeability, and the ability to form ion channels upon reconstitution in planar membranes. This compound showed no signs of cross resistance to targets of current clinical candidates, and 3 independent lines evolved to resist HZ NPDG-I acquired nonsynonymous changes in the P. falciparum multidrug resistance transporter, pfmdr1. Conditional knockdown of PfMDR1 showed varying effects to other peptaibol analogs, suggesting differing sensitivity.

Pseudodiploöspora longispora (previously known as Diploöspora longispora) is a pathogenic fungus of Morchella mushrooms. The molecular mechanism underlying the infection of P. longispora in fruiting bodies remains unknown. In this study, three known peptaibols, alamethicin F-50, polysporin B, and septocylindrin B (1-3), and a new analogue, longisporin A (4), were detected and identified in the culture of P. longispora and the fruiting bodies of M. sextelata infected by P. longispora. The primary amino sequence of longisporin A is defined as Ac-Aib1-Pro2-Aib3-Ala4-Aib5-Aib6-Gln7-Aib8-Val9-Aib10-Glu11-Leu12-Aib13-Pro14-Val15-Aib16-Aib17-Gln18-Gln19-Phaol20. The peptaibols 1-4 greatly suppressed the mycelial growth of M. sextelata. In addition, treatment with alamethicin F-50 produced damage on the cell wall and membrane of M. sextelata. Compounds 1-4 also exhibited inhibitory activities against human pathogens including Aspergillus fumigatus, Candida albicans, methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus, and plant pathogen Verticillium dahlia. Herein, peptaibols are confirmed as virulence factors involved in the invasion of P. longispora on Morchella, providing insights into the interaction between pathogenic P. longispora and mushrooms.