FMOe: Preprocessing and Visualizing Package of the Fragment Molecular Orbital Method for Molecular Operating Environment and Its Applications in Covalent Ligand and Metalloprotein Analyses.

The fragment molecular orbital (FMO) method is an efficient quantum chemical calculation technique for large biomolecules, dividing each into smaller fragments and providing interfragment interaction energies (IFIEs) that support our understanding of molecular recognition. The ab initio fragment MO method (ABINIT-MP), an FMO processing program, can automatically divide typical proteins and nucleic acids. In contrast, small molecules such as ligands and heterosystems must be manually divided. Thus, we developed a graphical user interface to easily handle such manual fragmentation as a library for the Molecular Operating Environment (MOE) that preprocesses and visualizes FMO calculations. We demonstrated fragmentation with IFIE analyses for the two following cases: (1) covalent cysteine-ligand bonding inside the SARS-CoV-2 main protease (Mpro) and nirmatrelvir (Paxlovid) complex and (2) the metal coordination inside a zinc-bound cyclic peptide. IFIE analysis successfully identified the key amino acid residues for the molecular recognition of nirmatrelvir with Mpro and the details of their interactions (e.g., hydrogen bonds and CH/π interactions) via ligand fragmentation of functional group units. In metalloproteins, we found an efficient and accurate scheme for the fragmentation of Zn2+ ions with four histidines coordinated to the ion. FMOe simplifies manual fragmentation, allowing users to experiment with various fragmentation patterns and perform in-depth IFIE analysis with high accuracy. In the future, our findings will provide valuable insight into complicated cases, such as ligand fragmentation in modality drug discovery, especially for medium-sized molecules and metalloprotein fragmentation around metals.

Triscysteine disulfide-directing motifs enabling design and discovery of multicyclic peptide binders.

Peptides are valuable for therapeutic development, with multicyclic peptides showing promise in mimicking antigen-binding potency of antibodies. However, our capability to engineer multicyclic peptide scaffolds, particularly for the construction of large combinatorial libraries, is still limited. Here, we study the interplay of disulfide pairing between three biscysteine motifs, and designed a range of triscysteine motifs with unique disulfide-directing capability for regulating the oxidative folding of multicyclic peptides. We demonstrate that incorporating these motifs into random sequences allows the design of disulfide-directed multicyclic peptide (DDMP) libraries with up to four disulfide bonds, which have been applied for the successful discovery of peptide binders with nanomolar affinity to several challenging targets. This study encourages the use of more diverse disulfide-directing motifs for creating multicyclic peptide libraries and opens an avenue for discovering functional peptides in sequence and structural space beyond existing peptide scaffolds, potentially advancing the field of peptide drug discovery.

Identification of the Vulnerability of Atherosclerotic Plaques by a Photoacoustic/Ultrasonic Dual-Modal cRGD Nanomolecular Probe.

Objective: To explore the feasibility of using cRGD-GNR-PFP-NPs to assess plaque vulnerability in an atherosclerotic plaque mouse model by dual-modal photoacoustic/ultrasonic imaging. Methods: A nanomolecular probe containing gold nanorods (GNRs) and perfluoropentane (PFP) coated with the cyclic Arg-Gly-Asp (cRGD) peptide were prepared by double emulsion solvent evaporation and carbodiimide methods. The morphology, particle size, potential, cRGD conjugation and absorption features of the nanomolecular probe were characterized, along with its in vitro phase transformation and photoacoustic/ultrasonic dual-modal imaging properties. In vivo fluorescence imaging was used to determine the distribution of cRGD-GNR-PFP-NPs in vivo in apolipoprotein E-deficient (ApoE-/-) atherosclerotic plaque model mice, the optimal imaging time was determined, and photoacoustic/ultrasonic dual-modal molecular imaging of integrin αvß3 expressed in atherosclerotic plaques was performed. Pathological assessments verified the imaging results in terms of integrin αvß3 expression and plaque vulnerability. Results: cRGD-GNR-PFP-NPs were spherical with an appropriate particle size (average of approximately 258.03±6.75 nm), a uniform dispersion, and a potential of approximately -9.36±0.53 mV. The probe had a characteristic absorption peak at 780~790 nm, and the surface conjugation of the cRGD peptide reached 92.79%. cRGD-GNR-PFP-NPs were very stable in the non-excited state but very easily underwent phase transformation under low-intensity focused ultrasound (LIFU) and had excellent photoacoustic/ultrasonic dual-modal imaging capability. Mice fed a high-fat diet for 20 weeks had obvious hyperlipidemia with larger, more vulnerable plaques. These plaques could be specifically targeted by cRGD-GNR-PFP-NPs as determined by in vivo fluorescence imaging, and the enrichment of nanomolecular probe increased with the increasing of plaque vulnerability; the photoacoustic/ultrasound signals of the plaques in the high-fat group were stronger. The pathological assessments were in good agreement with the cRGD-GNR-PFP-NPs plaque accumulation, integrin αvß3 expression and plaque vulnerability results. Conclusion: A phase variant photoacoustic/ultrasonic dual-modal cRGD nanomolecular probe was successfully prepared and can be used to identify plaque vulnerability safely and effectively.

Nanoencapsulation enhances the antimicrobial and antioxidant stability of cyclic lipopeptides for controlling Fusarium graminearum.

Fusarium graminearum not only causes Fusarium head blight (FHB) on wheat but also produces fungal toxins that pose a serious threat to food safety. Biological control is one of the safe and most effective alternative methods. In this study, cyclic lipopeptides (CLPs) produced from Bacillus mojavensis B1302 were extracted and identified by LC-MS/MS. After preparing mesoporous silica nanoparticles-NH2 (MSNsN) and encapsulating CLPs, the characterization analysis showed that the interaction between CLPs and MSNsN enhanced the crystal structure of CLPs-MSNsN. The antimicrobial activity and antioxidant capacity of CLPs-MSNsN stored at 20 °C and 45 °C were decreased more slowly than those of free CLPs with increasing storage time, indicating the enhancement of the antimicrobial and antioxidant stability of CLPs. Moreover, the field control efficacy of long-term stored CLPs-MSNsN only decreased from 78.66% to 63.2%, but the efficacy of free CLPs decreased significantly from 84.34% to 26.01%. The deoxynivalenol (DON) content of wheat grains in the CLPs-MSNsN treatment group was lower than that in the free CLPs treatment group, which showed that long-term stored CLPs-MSNsN reduced the DON content in wheat grains. Further analysis of the action mechanism of CLPs-MSNsN on F. graminearum showed that CLPs-MSNsN could disrupt mycelial morphology, cause cell apoptosis, lead to the leakage of proteins and nucleic acids, and destroy the cell permeability of mycelia. This work puts a novel insight into the antimicrobial and antioxidant stability enhancement of CLPs-MSNsN through encapsulation and provides a potential fungicide to control F. graminearum, reduce toxins and ensure food safety.

The n-Butanol Extract Obtained from the Inner Bark of Tabebuia rosea (Bertol.) DC, Specioside, and Catalposide Induce Leukemia Cell Apoptosis in the Presence of Apicidin.

The cell signaling pathways involved in the antiproliferative activities of T. rosea inner bark remain unexplored. This study evaluated the apoptotic effects of two iridoids from the inner bark of T. rosea and apicidin on THP-1 cells. The cytotoxic effects of the extract and the pure compounds on THP-1 and Jurkat cells were also evaluated using the MTT assay. The apoptotic effect was determined by measuring the mitochondrial membrane potential. The expression of mRNA and MAPK kinase, Bax, and Bcl-2 proteins was detected by Western blotting and RT-qPCR, respectively. The extract and the compounds evaluated increased the percentage of apoptotic cells. Depolarization of the mitochondrial membrane was observed, and the number of cells in the G0/G1 phase increased. Catalposide and specioside significantly increased p38 protein expression, mostly in cells pretreated with apicidin. The p38 MAPK signaling pathway is at least one of the pathways by which the n-butanol extract obtained from Tabebuia rosea, catalposide, and specioside exerts its apoptotic effect on THP-1 cells, and this effect generates a response in the G0/G1 phase and subsequent cell death. In addition, there was depolarization of the mitochondrial membrane, an effect that was related to the participation of the proapoptotic protein Bax.

Structure Derivatization of IgG-Binding Peptides and Analysis of Their Secondary Structure by Circular Dichroism Spectroscopy.

Mid-sized cyclic peptides are a promising modality for modern drug discovery. Their larger interaction area coupled with an appropriate secondary structure is more suitable than small molecules for binding to the target protein. In this study, we conducted a structure derivatization of an immunoglobulin G (IgG)-binding peptide (15-IgBP), a ß-hairpin-like cyclic peptide with a twisted ß-strand and assessed the effect of the secondary structure on IgG-binding activity using circular dichroism (CD) spectra analysis. As a result, derivatization at the Ala5 and Gly9 positions affected the secondary structure of 15-IgBP, in particular the appearance of a small positive peak in the 220-240 nm region characteristic of 15-IgBP in the CD spectrum. Maintaining this peak at a moderate level may be important for the expression of IgG binding activity. We found the small methyl group at Ala5 to be crucial for retaining the preferred secondary structure; we also found Gly9 could be replaced by D-amino acids. By integrating these findings with previous results of the structure-activity relationship, we obtained four potent affinity peptides for IgG binding (Kd = 4.24-5.85 nM). Furthermore, we found the Gly9 position can be substituted for D-Lys. This is a new potential site for attaching functional units for conjugation with IgG for the preparation of homogeneous antibody-drug conjugates.

Synthesis and Cytotoxicity of Cyclic Octapeptide Surugamides with Varied N-Acyl Moieties.

Surugamides are a group of non-ribosomal peptides produced by Streptomyces spp. Several derivatives possess acyl groups, which are proposed to be attached to a lysine side chain after backbone-macrocyclization during biosynthesis. To date, five different acyl groups have been identified in nature, yet their impacts on biological activity remain underexplored. Here we synthesized surugamide B derivatives with varied acyl moieties. Biological evaluations revealed that larger hydrophobic acyl groups on lysine ε-NH2 enhance cytotoxicity.

Development and preclinical characterization of a novel radiotheranostic EphA2-targeting bicyclic peptide.

Erythropoietin-producing hepatocellular receptor A2 (EphA2), is a receptor tyrosine kinase involved in cell-cell interactions. It is known to be overexpressed in various tumors and is associated with poor prognosis. EphA2 has been proposed as a target for theranostic applications. Low molecular weight peptide-based scaffolds with low nanomolar affinities have been shown to be ideal in such applications. Bicyclic peptides have emerged as an alternative to traditional peptides for this purpose, offering affinities comparable to antibodies due to their constrained nature, along with high tissue penetration, and improved stability compared to linear counterparts. This study presents the development and comprehensive in vitro and in vivo preclinical evaluation of BCY18469, a novel EphA2-targeting bicyclic peptide-based radiotheranostic agent. Methods: The EphA2-targeting Bicycle® peptide BCY18469 was identified through phage-display and chemically optimized. BCY18469 was radiolabeled with 68Ga, 177Lu and 111In. The physicochemical properties, binding affinity and internalization as well as specificity of the peptide were evaluated in vitro. In vivo PET/MR and SPECT/CT imaging studies were performed using [68Ga]Ga-BCY18469 and [111In]In-BCY18469, respectively, along with biodistribution of [177Lu]Lu-BCY18469 up to 24 h post injection in HT1080- and PC-3-tumor bearing BALB/c nu/nu EphA2-overexpressing xenograft mouse models. Results: The EphA2-targeting bicyclic peptide BCY18469 showed high binding affinity toward human and mouse EphA2 (1.9 and 3.8 nM, respectively). BCY18469 specifically bound and internalized into EphA2-expressing HT1080 cells. Imaging studies showed high tumor enrichment at early time-points (SUV of 1.7 g/mL at 1 h p.i. and 1.2 g/mL at 2 h p.i. in PET/MRI, HT1080 xenograft) with tumor contrast as early as 5 min p.i. and kidney-mediated clearance. Biodistribution studies revealed high early tumor uptake (19.5 ± 3.5 %ID/g at 1 h p.i., HT1080 xenograft) with SPECT/CT imaging further confirming these findings (5.7 ± 1.5 %ID/g at 1 h p.i., PC-3 xenograft). Conclusion: BCY18469 demonstrated high affinity, specific targeting of EphA2, a favorable biodistribution profile, and clearance through renal pathways. These findings underscore the potentially important role of bicyclic peptides in advancing radiotheranostic approaches and encourage additional translational research.

Determinants of maturation of the Staphylococcus aureus autoinducing peptide.

The accessory gene regulatory (Agr) system is required for virulence factor gene expression and pathogenesis of Staphylococcus aureus. The Agr system is activated in response to the accumulation of a cyclic autoinducing peptide (AIP), which is matured and secreted by the bacterium. The precursor of AIP, AgrD, consists of the AIP flanked by an N-terminal [Formula: see text]-helical Leader and a charged C-terminal tail. AgrD is matured to AIP by the action of two proteases, AgrB and MroQ. AgrB cleaves the C-terminal tail and promotes the formation of a thiolactone ring, whereas MroQ cleaves the N-terminal Leader in a manner that depends on the four-amino acid linker immediately following a conserved IG helix breaker motif. However, the attributes of AgrD that dictate the sequence of events in peptide maturation are not fully defined. Here, we used engineered AgrD peptide intermediates to ascertain the sufficiency of MroQ for N-terminal peptide cleavage, peptide export, and generation of mature AIP. We found that MroQ promotes the removal of the N-terminal Leader peptide from both linear and cyclic peptide intermediates, while peptide cyclization remained essential for signaling. The expression of the Leader peptide in isolation was sufficient for MroQ-dependent cleavage proximal to the four-amino-acid linker. In addition, active site mutations within AgrB destabilized full-length AgrD and thiolactone-containing intermediates and prevented the release of the Leader peptide. Altogether, our data support a tandem peptide maturation event involving both MroQ and AgrB that appears to couple protease activity and export of bioactive AIP.IMPORTANCEThe accessory gene regulatory (Agr) system is important for S. aureus pathogenesis. Activation of the Agr system requires recognition of a cyclic peptide pheromone, which must be fully matured to exert its biological activity. The complete events in cyclic peptide maturation and export from the bacterial cell remain to be fully defined. We and others recently discovered that the membrane peptidase MroQ is required for pheromone maturation. This study builds off the identification of MroQ and considers the attributes of the pheromone pro-peptide that are required for MroQ-mediated processing as well as uncovers features important for peptide stability and export. Overall, the findings in this study have implications for understanding bacterial pheromone maturation and virulence.

Heterologous Expression Facilitates the Production and Characterization of a Class III Lanthipeptide with Coupled Labionin Cross-Links in Sponge-Associated Streptomyces rochei MB037.

Cyclic peptides, with remarkable stability, cellular permeability, and proteolysis resistance, display promising potential in pharmaceutical applications. Labionin (Lab), a unique bicyclic cross-link containing both C-C and C-S bonds, provides high rigidity and better control of conformation compared to monocyclic cross-links. To discover more Lab-containing scaffolds with highly rigid conformation for cyclic peptide drug development, herein, a cryptic class III lanthipeptide biosynthetic gene cluster (BGC) (i.e., rcs) was identified in the sponge-associated Streptomyces rochei MB037 and expressed in Escherichia coli, incorporating an N-terminal SUMO-tag on the RcsA precursor peptide to prevent proteolysis. Subsequently, a novel class III lanthipeptide, i.e., rochsin A, exhibiting a highly rigid conformation with coupled Lab cross-links crowded by bulky aromatic amino acids, was produced. Three AplP-like proteases outside the rcs BGC were proven to remove the leader peptide of rochsin A through their dual endo- and aminopeptidase activities, resulting in mature rochsin A in vitro. Ala mutation experiments revealed the C to N cyclization direction, like most class III lanthipeptides. However, RcsKC displays a high substrate breadth, enabling various ring topologies that are rarely observed in other class III lanthipeptides. Overall, the established expression system broadens the chemical diversity of cyclic peptides with unique Lab cross-links and offers a highly rigid scaffold for cyclic peptide drug development.

Near-infrared pH-switchable BODIPY photosensitizers for dual biotin/cRGD targeted photodynamic therapy.

Photodynamic therapy (PDT) is a clinically-approved cancer treatment that is based on production of cytotoxic reactive oxygen species to induce cell death. However, its efficiency depends on distribution of photosensitizer (PS) and depth of light penetration through the tissues. Tendency of pathological cancer tissues to exhibit lower pH than healthy tissues inspired us to explore dual-targeted pH-activatable photosensitizers based on tunable near-infrared (NIR) boron-dipyrromethene (BODIPY) dyes. Our BODIPY PSs were designed to carry three main attributes: (i) biotin or cRGD peptide as an effective cancer cell targeting unit, (ii) amino moiety that is protonated in acidic (pH <6.5) conditions for pH-activation of the PS based on photoinduced electron transfer (PET) and (iii) hydrophilic groups enhancing the water solubility of very hydrophobic BODIPY dyes. Illumination of such compounds with suitable light (>640nm) allowed for high phototoxicity against HeLa (αvß3 integrin and biotin receptor positive) and A549 (biotin receptor positive) cells compared to healthy MRC-5 (biotin negative) cells. Moreover, no dark toxicity was observed on selected cell lines (>10 µM) providing promising photosensitizers for tumour-targeted photodynamic therapy.

Sickle Cell Hemoglobin "Drugged" with Cyclic Peptides Is Aggregation Incompetent.

Sickle cell disease (SCD) is a monogenic blood disorder associated with a mutation in the hemoglobin subunit ß gene encoding for the ß-globin of normal adult hemoglobin (HbA). This mutation transcribes into a Glu-ß6 → Val-ß6 substitution in the ß-globins, inducing the polymerization of this hemoglobin form (HbS) when in the T-state. Despite advances in stem cell and gene therapy, and the recent approval of a new antisickling drug, therapeutic limitations persist. Herein, we demonstrate through molecular dynamics and umbrella sampling, that (unrestrained) blockage of the hydrophobic pocket involved in the lateral contact of the HbS fibers by 5-mer cyclic peptides, recently proposed as SCD aggregation inhibitors (Neto, V.; J. Med. Chem. 2023, 66, 16062-16074), is enough to turn the dimerization of HbS thermodynamically unfavorable. Among these potential drugs, some exhibit an estimated pocket abandonment probability of around 15-20% within the simulations' time frame, and an impressive specificity toward the mutated Val-ß6. Additionally, we show that the dimerization can be thermodynamically unfavored by blocking a nearby region while the pocket remains vacant. These results are compared with curcumin, an antisickling molecule and a pan-assay interference compound, with a good binding affinity for different proteins and protein domains. Our results confirm the potential of some of these cyclic peptides as antisickling drug candidates to reduce the concentration of aggregation-competent HbS.

CycPeptMP: enhancing membrane permeability prediction of cyclic peptides with multi-level molecular features and data augmentation.

Cyclic peptides are versatile therapeutic agents that boast high binding affinity, minimal toxicity, and the potential to engage challenging protein targets. However, the pharmaceutical utility of cyclic peptides is limited by their low membrane permeability-an essential indicator of oral bioavailability and intracellular targeting. Current machine learning-based models of cyclic peptide permeability show variable performance owing to the limitations of experimental data. Furthermore, these methods use features derived from the whole molecule that have traditionally been used to predict small molecules and ignore the unique structural properties of cyclic peptides. This study presents CycPeptMP: an accurate and efficient method to predict cyclic peptide membrane permeability. We designed features for cyclic peptides at the atom-, monomer-, and peptide-levels and seamlessly integrated these into a fusion model using deep learning technology. Additionally, we applied various data augmentation techniques to enhance model training efficiency using the latest data. The fusion model exhibited excellent prediction performance for the logarithm of permeability, with a mean absolute error of $0.355$ and correlation coefficient of $0.883$. Ablation studies demonstrated that all feature levels contributed and were relatively essential to predicting membrane permeability, confirming the effectiveness of augmentation to improve prediction accuracy. A comparison with a molecular dynamics-based method showed that CycPeptMP accurately predicted peptide permeability, which is otherwise difficult to predict using simulations.

CREMP: Conformer-rotamer ensembles of macrocyclic peptides for machine learning.

Computational and machine learning approaches to model the conformational landscape of macrocyclic peptides have the potential to enable rational design and optimization. However, accurate, fast, and scalable methods for modeling macrocycle geometries remain elusive. Recent deep learning approaches have significantly accelerated protein structure prediction and the generation of small-molecule conformational ensembles, yet similar progress has not been made for macrocyclic peptides due to their unique properties. Here, we introduce CREMP, a resource generated for the rapid development and evaluation of machine learning models for macrocyclic peptides. CREMP contains 36,198 unique macrocyclic peptides and their high-quality structural ensembles generated using the Conformer-Rotamer Ensemble Sampling Tool (CREST). Altogether, this new dataset contains nearly 31.3 million unique macrocycle geometries, each annotated with energies derived from semi-empirical extended tight-binding (xTB) DFT calculations. Additionally, we include 3,258 macrocycles with reported passive permeability data to couple conformational ensembles to experiment. We anticipate that this dataset will enable the development of machine learning models that can improve peptide design and optimization for novel therapeutics.

A short-lived peptide signal regulates cell-to-cell communication in Listeria monocytogenes.

Quorum sensing (QS) is a mechanism that regulates group behavior in bacteria, and in Gram-positive bacteria, the communication molecules are often cyclic peptides, called autoinducing peptides (AIPs). We recently showed that pentameric thiolactone-containing AIPs from Listeria monocytogenes, and from other species, spontaneously undergo rapid rearrangement to homodetic cyclopeptides, which hampers our ability to study the activity of these short-lived compounds. Here, we developed chemically modified analogues that closely mimic the native AIPs while remaining structurally intact, by introducing N-methylation or thioester-to-thioether substitutions. The stabilized AIP analogues exhibit strong QS agonism in L. monocytogenes and allow structure-activity relationships to be studied. Our data provide evidence to suggest that the most potent AIP is in fact the very short-lived thiolactone-containing pentamer. Further, we find that the QS system in L. monocytogenes is more promiscuous with respect to the structural diversity allowed for agonistic AIPs than reported for the more extensively studied QS systems in Staphylococcus aureus and Staphylococcus epidermidis. The developed compounds will be important for uncovering the biology of L. monocytogenes, and the design principles should be broadly applicable to the study of AIPs in other species.

(S)-3-(3,4-Dihydroxybenzyl) piperazine-2,5-dione (cyclo-Gly-L-DOPA or CG-Nio-CGLD) peptide loaded in Chitosan Glutamate-Coated Niosomes as anti-Colorectal cancer activity.

BACKGROUND: Colorectal cancer (CRC), now the second most prevalent malignant tumor worldwide, is more prevalent in young adults. In recent decades, there has been progress in creating anti-colorectal cancer medications, including cytotoxic compounds. OBJECTIVES: Novel anticancer drugs are needed to surmount existing obstacles. A recent study investigated the effectiveness of novel formulations in preventing colorectal cancer. METHODS: During this study, we assessed a new kind of niosome called cyclo-Gly-L-DOPA (CG-Nio-CGLD) made from chitosan glutamate. We evaluated the anti-colorectal cancer properties of CG-Nio-CGLD utilizing CCK-8, invasion assay, MTT assay, flow cytometry, and cell cycle analysis. The transcription of genes associated with apoptosis was analyzed using quantitative real-time PCR. At the same time, the cytotoxicity of nanomaterials on both cancer and normal cell lines was assessed using MTT assays. Novel anticancer drugs are needed to surmount existing obstacles. A recent study investigated the effectiveness of newly developed formulations in preventing colorectal cancer. RESULTS: The Nio-CGLD and CG-Nio-CGLD were spherical mean diameters of 169.12 ± 1.87 and 179.26 ± 2.17 nm, respectively. Entrapment efficiency (EE%) measurements of the Nio-CGLD and CG-Nio-CGLD were 63.12 ± 0.51 and 76.43 ± 0.34%, respectively. In the CG-Nio-CGLD group, the percentages of early, late, necrotic, and viable CL40 cells were 341.93%, 23.27%, 9.32%, and 25.48%. The transcription of the genes PP53, cas3, and cas8 was noticeably higher in the treatment group compared to the control group (P > 0.001). Additionally, the treatment group had lower BCL2 and survivin gene expression levels than the control group (P < 0.01). Additionally, CG-Nio-CGLD formulations demonstrated a biocompatible nanoscale delivery mechanism and displayed little cytotoxicity toward the CCD 841 CoN reference cell line. CONCLUSION: These findings indicate that chitosan-based noisome encapsulation may enhance the effectiveness of CG-Nio-CGLD formulations in fighting cancer.

Insights into the antifungal mechanism of Bacillus subtilis cyclic lipopeptide iturin A mediated by potassium ion channel.

Fungal infections pose severe and potentially lethal threats to plant, animal, and human health. Ergosterol has served as the primary target for developing antifungal medications. However, many antifungal drugs remain highly toxic to humans due to similarity in cell membrane composition between fungal and animal cells. Iturin A, lipopeptide produced by Bacillus subtilis, efficiently inhibit various fungi, but demonstrated safety in oral administration, indicating the existence of targets different from ergosterol. To pinpoint the exact antifungal target of iturin A, we used homologous recombination to knock out and overexpress erg3, a key gene in ergosterol synthesis. Saccharomyces cerevisiae and Aspergillus carbonarius were transformed using the LiAc/SS-DNNPEG and Agrobacterium-mediated transformation (AMT), respectively. Surprisingly, increasing ergosterol content did not augment antifungal activity. Furthermore, iturin A's antifungal activity against S. cerevisiae was reduced while it pre-incubation with voltage-gated potassium (Kv) channel inhibitor, indicating that Kv activation was responsible for cell death. Iturin A was found to activate the Kv protein, stimulating K+ efflux from cell. In vitro tests confirmed interaction between iturin A and Kv protein. This study highlights Kv as one of the precise targets of iturin A in its antifungal activity, offering a novel target for the development of antifungal medications.

An N-ortho-nitrobenzylated benzanilide amino acid enables control of the conformation and membrane permeability of cyclic peptides.

We designed and synthesized an N-ortho-nitrobenzylated benzanilide-based amino acid having a cis-amide structure that facilitates cyclization of peptides containing it. Photo-induced removal of the nitrobenzyl group from this residue in the resulting cyclized peptides dramatically alters their conformation and passive membrane permeability via complete cis-amide to trans-amide conversion.

Discovery of Thioether-Cyclized Macrocyclic Covalent Inhibitors by mRNA Display.

Macrocyclic peptides are promising scaffolds for the covalent ligand discovery. However, platforms enabling the direct identification of covalent macrocyclic ligands in a high-throughput manner are limited. In this study, we present an mRNA display platform allowing selection of covalent macrocyclic inhibitors using 1,3-dibromoacetone-vinyl sulfone (DBA-VS). Testcase selections on TEV protease resulted in potent covalent inhibitors with diverse cyclic structures, among which cTEV6-2, a macrocyclic peptide with a unique C-terminal cyclization, emerged as the most potent covalent inhibitor of TEV protease described to-date. This study outlines the workflow for integrating chemical functionalization─installation of a covalent warhead─with mRNA display and showcases its application in targeted covalent ligand discovery.

Oxidation-guided and collision-induced linearization assists de novo sequencing of thioether macrocyclic peptides.

Oxidation of a thioether linkage in thioether-closed macrocyclic peptides led to collision-induced site-selective linearization of the peptides. This method has allowed for de novo sequencing of thioether macrocyclic peptides. The utility of the sequencing method was demonstrated by identifying the correct peptide sequences from a virtually randomized thioether macrocyclic peptide library.