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.

Trichorzin PA is a family of 18-residue peptaibols with linear and flexible peptide chains. The three-dimensional structures and biological activities of trichorzin PA peptaibols are largely uncharacterised. In this work, two previously identified peptaibols, trichorzin PA VI (1) and II (2), were isolated from Trichoderma lentiforme ML-P8-2. While for the first time, we report here the X-ray crystallographic structure of 1, antimicrobial activities against a panel of common pathogenic bacteria and fungi, and cytotoxicities of 1 and 2. In bioassays, 1 and 2 exhibited strong antimicrobial activities against the seven tested microbes, with MIC values in the range of 0.19-6.25 μM. Additionally, 1 and 2 displayed potent cytotoxicities against five human cancer cell lines, with IC50 values in the range of 0.01 ± 0.02-2.75 ± 0.17 μM. The bioassay results were generally better than those reported for other 18-residue peptaibols, including other trichorzin PA members.

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.

The oral pathogen Fusobacterium nucleatum has recently been associated with an elevated risk of colorectal cancer (CRC), endometrial metastasis, chemoresistance, inflammation, metastasis, and DNA damage, along with several other diseases. This study aimed to explore the disruption of protein machinery of F. nucleatum via inhibition of elongation factor thermo unstable (Ef-Tu) protein, through natural products. No study on Ef-Tu inhibition by natural products or in Fusobacterium spp. exists till todate. Ef-Tu is an abundant specialized drug target in bacteria that varies from human Ef-Tu. Elfamycins target Ef-Tu and hence, Enacyloxin IIa was used to generate pharmacophore for virtual screening of three natural product libraries, Natural Product Activity and Species Source (NPASS) (n = 30000 molecules), Tibetan medicinal plant database (n = 54 molecules) and African medicinal plant database (n > 6000 molecules). Peptaibol Septocylindrin B (NPC141050), Hirtusneanoside, and ZINC95486259 were prioritized from these libraries as potential therapeutic candidates. ADMET profiling was done for safety assessment, physiological-based pharmacokinetic modeling in human and mouse for getting insight into drug interaction with body tissues and molecular dynamics was used to assess stability of the best hit NPC141050 (Septocylindrin B). Based on the promising results, we propose further in vitro, in vivo and pharmacokinetic testing on the lead Septocylindrin B, for possible translation into therapeutic interventions.

In an effort to identify novel compounds with potent inhibition against Toxoplasma gondii, a phenotypic screen was performed utilizing a library of 683 pure compounds derived primarily from terrestrial and marine fungi. An initial screen with a fixed concentration of 5 µM yielded 91 hits with inhibition comparable to an equal concentration of artemisinin. These compounds were then triaged based on known biological and chemical concerns and liabilities. From these, 49 prioritized compounds were tested in a dose response format with T. gondii and human foreskin fibroblasts (HFFs) for cytotoxicity. Ten compounds were identified with an IC50 less than 150 nM and a selectivity index (SI) greater than 100. An additional eight compounds demonstrated submicromolar IC50 and SI values equal to or greater than 35. While the majority of these scaffolds have been previously implicated against apicomplexan parasites, their activities in T. gondii were largely unknown. Herein, we report the T. gondii activity of these compounds with chemotypes including xanthoquinodins, peptaibols, heptelidic acid analogs, and fumagillin analogs, with multiple compounds demonstrating exceptional potency in T. gondii and limited toxicity to HFFs at the highest concentrations tested.
OBJECTIVE: Current therapeutics for treating toxoplasmosis remain insufficient, demonstrating high cytotoxicity, poor bioavailability, limited efficacy, and drug resistance. Additional research is needed to develop novel compounds with high efficacy and low cytotoxicity. The success of artemisinin and other natural products in treating malaria highlights the potential of natural products as anti-protozoan therapeutics. However, the exploration of natural products in T. gondii drug discovery has been less comprehensive, leaving untapped potential. By leveraging the resources available for the malaria drug discovery campaign, we conducted a phenotypic screen utilizing a set of natural products previously screened against Plasmodium falciparum. Our study revealed 18 compounds with high potency and low cytotoxicity in T. gondii, including four novel scaffolds with no previously reported activity in T. gondii. These new scaffolds may serve as starting points for the development of toxoplasmosis therapeutics but could also serve as tool compounds for target identification studies using chemogenomic approach.

We previously reported on a novel peptaibol, named Tripleurin XIIc (TPN), an 18-residue long sequence produced by the fungus Trichoderma pleuroti. We elucidated its 3D structure via classical and accelerated molecular dynamics simulation (aMD) methods and reported the folding dynamics of TPN in water and chloroform solvents. Peptaibols, in general, are insoluble in water, as they are amphipathic and may prefer hydrophobic environments like transmembrane regions. In this study, we attempted to use aMD simulations to model an all-atom bacterial membrane system while placing a TPN molecule in its vicinity. The results highlighted that TPN was able to introduce some disorder into the membrane and caused lipid clustering. It could also enter the transmembrane region from the water-bilayer interface. The structural dynamics of TPN in the transmembrane region revealed a single energetically stable conformation similar to the one obtained from water and chloroform solvent simulations reported by us previously. However, this linear structure was found to be at the local energy minimum (stable) in water but at a metastable intermediate state (higher energy) in chloroform. Therefore, it could be said that the water solvent can be successfully used for folding simulations of peptaibols.

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.

Peptaibols are proteolysis-resistant, membrane-active peptides. Their remarkably stable helical 3D-structures are key for their bioactivity. They can insert themselves into the lipid bilayer as barrel staves, or lay on its surface like carpets, depending on both their length and the thickness of the lipid bilayer. Medium-length peptaibols are of particular interest for studying the peptide-membrane interaction because their length allows them to adopt either orientation as a function of the membrane thickness, which, in turn, might even result in an enhanced selectivity. Electron paramagnetic resonance (EPR) is the election technique used to this aim, but it requires the synthesis of spin-labeled medium-length peptaibols, which, in turn, is hampered by the poor reactivity of the Cα-tetrasubstituted residues featured in their sequences. After several years of trial and error, we are now able to give state-of-the-art advice for a successful synthesis of nitroxide-containing peptaibols, avoiding deleted sequences, side reactions and difficult purification steps. Herein, we describe our strategy and itsapplication to the synthesis of spin-labeled analogs of the recently discovered, natural, medium-length peptaibol pentadecaibin. We studied the antitumor activity of pentadecaibin and its analogs, finding potent cytotoxicity against human triple-negative breast cancer and ovarian cancer. Finally, our analysis of the peptide conformational preferences and membrane interaction proved that pentadecaibinspin-labeling does not alter the biological features of the native sequence and is suitable for further EPR studies. The nitroxide-containing pentadecaibins, and their synthetic strategy described herein, will help to shed light on the mechanism of the peptide-membrane interaction of medium-length peptaibols.

New classes of antibacterial drugs are urgently needed to address the global issue of antibiotic resistance. In this context, peptaibols are promising membrane-active peptides since they are not involved in innate immunity and their antimicrobial activity does not involve specific cellular targets, therefore reducing the chance of bacterial resistance development. Trichogin GA IV is a nonhemolytic, natural, short-length peptaibol active against Gram-positive bacteria and resistant to proteolysis. In this work, we report on the antibacterial activity of cationic trichogin analogs. Several peptides appear non-hemolytic and strongly active against many clinically relevant bacterial species, including antibiotic-resistant clinical isolates, such as Staphylococcus aureus, Acinetobacter baumannii, and extensively drug-resistant Pseudomonas aeruginosa, against which there are only a limited number of antibiotics under development. Our results further highlight how the modification of natural peptides is a valuable strategy for obtaining improved antibacterial agents with potential therapeutic applications.

In the large field of bioactive peptides, peptaibols represent a unique class of compounds. They are membrane-active peptides, produced by fungi of the genus Trichoderma and known to elicit plant defenses. Among the short-length peptaibols, trichogin GA IV is nonhemolytic, proteolysis-resistant, antibacterial, and cytotoxic. Several trichogin analogs are endowed with potent activity against phytopathogens, thus representing a sustainable alternative to copper for plant protection. In this work, we tested the activity of trichogin analogs against a breast cancer cell line and a normal cell line of the same derivation. Lys-containing trichogins showed an IC50 below 12 µM, a peptide concentration not significantly affecting the viability of normal cells. Two analogs were found to be membrane-active but noncytotoxic. They were anchored to gold nanoparticles (GNPs) and further investigated for their ability to act as targeting agents. GNP uptake by cancer cells increased with peptide decoration, while it decreased in the corresponding normal epithelial cells. This work highlights the promising biological properties of peptaibol analogs in the field of cancer therapy either as cytotoxic molecules or as active targeting agents in drug delivery.