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

Abstract

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...

A genetics-free method for high-throughput discovery of cryptic microbial metabolites

“…LAESI-IMS has been employed for spatially resolving secondary metabolite production and for strain selection. 23,24,49 High-throughput elicitation has previously been reported in a compound- and cluster-specific manner along with ion-mobility UPLC-MS to assess a small number of eliciting conditions. 18,50,51 By combining the two overarching methods – high-throughput elicitation and IMS – HiTES-IMS facilitates interrogation of cryptic bacterial metabolomes in response to hundreds of conditions.…”

Monitoring the Site-Specific Solid-State NMR Data in Oligopeptides

“…One of the most important areas of the research aims at finding an alternative antimicrobial strategy [2]. Related host-defense oligopeptides [3], mostly of the peptaibol family [4], have been analyzed in terms of their function, which is based on an ability to form pores in biological membranes [5]. The solid-state nuclear magnetic resonance (SSNMR) measurements are crucial in understanding the pore formation properties of peptaibols [6].…”

Sphaerostilbellins, New Antimicrobial Aminolipopeptide Peptaibiotics from Sphaerostilbella toxica

“…Fragmentation of the [M + 2H] 2+ ion at m / z 893.5827 resulted in a series of observable b/y fragments, in accordance with the sequential loss of amino acid residues from the N- and C- termini, respectively. Interestingly, formation of specific pairs of b/y fragments derived from the α-cleavage of β-Ala ions were greatly suppressed or absent, a feature common in other peptaibiotics containing β-Ala residues [ 28 , 29 ], which further supported the existence and positions of the β-Ala units in the proposed amino acid sequence.…”