Therapeutic stapled peptides: Efficacy and molecular targets.

Peptide stapling, by employing a stable, preformed alpha-helical conformation, results in the production of peptides with improved membrane permeability and enhanced proteolytic stability, compared to the original peptides, and provides an effective solution to accelerate the rapid development of peptide drugs. Various reviews present peptide stapling chemistries, anchoring residues and one- or two-component cyclization, however, therapeutic stapled peptides have not been systematically summarized, especially focusing on various disease-related targets. This review highlights the latest advances in therapeutic peptide drug development facilitated by the application of stapling technology, including different stapling techniques, synthetic accessibility, applicability to biological targets, potential for solving biological problems, as well as the current status of development. Stapled peptides as therapeutic drug candidates have been classified and analysed mainly by receptor- and ligand-based stapled peptide design against various diseases, including cancer, infectious diseases, inflammation, and diabetes. This review is expected to provide a comprehensive reference for the rational design of stapled peptides for different diseases and targets to facilitate the development of therapeutic peptides with enhanced pharmacokinetic and biological properties.

Integrated Physiologic and Proteomic Analyses Reveal the Molecular Mechanism of Navicula sp. in Response to Ultraviolet Irradiation Stress.

With the continuous development of space station construction, space ecosystem research has attracted increasing attention. However, the complicated responses of different candidate plants and algae to radiation stress remain unclear. The present study, using integrated physiologic and proteomic analyses, was carried out to reveal the molecular mechanism of Navicula sp. in response to ultraviolet (UV) irradiation stress. Under 12~24 h of high-dose UV irradiation conditions, the contents of chlorophyll and soluble proteins in Navicula sp. cells were significantly higher than those in the control and 4~8 h of low-dose UV irradiation groups. The activity of catalase (CAT) increased with the extension of irradiation time, and the activity of superoxide dismutase (SOD) decreased first and then increased. Furthermore, differential volcano plot analysis of the proteomic data of Navicula sp. samples found only one protein with a significant difference. Differential protein GO analysis unveiled that UV irradiation can activate the antioxidant system of Navicula sp. and further impact photosynthesis by affecting the photoreaction and chlorophyll synthesis of Navicula sp. The most significant differences in KEGG pathway analysis were also associated with photosynthesis. The above results indicate that Navicula sp. has good UV radiation resistance ability by regulating its photosynthetic pigment content, photosynthetic activity, and antioxidant system, making it a potential candidate for the future development of space ecosystems.

Discovery of novel antibacterial agent for the infected wound treatment: all-hydrocarbon stapling optimization of LL-37.

Rationale: Antimicrobial peptide LL-37 has been recognized as a favorable alternative to antibiotics due to its broad antibacterial spectrum, low resistance development and diverse biological activities. However, its high manufactory cost, poor proteolytic stability, and unpredictable cytotoxicity seriously hindered its medical translation. Methods: To push the frontiers of its clinical application, all-hydrocarbon stapling strategy was exploited here for the structural modification of KR-12, the core and minimal fragment of LL-37. Results: Based on a library of KR-12 derivatives that designed and synthesized to be stapled at positions of either i, i+4 or i, i+7, structure to activity relationship was investigated. Among them, KR-12(Q5, D9) with the glutamine and aspartic acid residues stapled displayed increased helical content and positive charge. The reinforced α-helical conformation not only protected it from proteolytic hydrolysis but also improved its antibacterial efficacy via effective membrane perturbation and anti-inflammatory efficacy via compact LPS binding. Besides, the increased positive charge endowed it with an enhanced therapeutic index. On infected wound mouse model, it was demonstrated to eliminate bacteria and promote wound closure and regeneration effectively. Conclusion: Overall, the all-hydrocarbon stapling was proven to lay the foundation for the future development of antibacterial agents. KR-12(Q5, D9) could serve as a lead compound for the clinical treatment of bacterial infections.

Cyclic peptide conjugated photosensitizer for targeted phototheranostics of gram-negative bacterial infection.

Antimicrobial photodynamic therapy (PDT) is a promising alternative to antibiotics for eradicating pathogenic bacterial infections. It holds advantage of not inducing antimicrobial resistance but is limited for the treatment of gram-negative bacterial infection due to the lack of photosensitizer (PS) capable of targeted permeating the outer membrane (OM) of gram-negative bacteria. To facilitate the targeted permeability of PS, cyclic polymyxin b nonapeptide that can specifically bind to the lipopolysaccharide on OM, is conjugated to an FDA approved PS chlorin e6 via variable linkers. Based on structure to activity study, C6pCe6 with aminohexanoic linker and P2pCe6 with amino-3, 6-dioxaoctanoic linker are identified to preferentially image gram-negative bacteria. These two conjugates also exhibit improved aqueous dispersity and enhanced ROS generation, consequently enabled their selective bactericidal activities against gram-negative bacteria upon 660 nm light irradiation. The effective photobactericidal ability of P2pCe6 is further validated on P. aeruginosa infected G. mellonella. Moreover, it is demonstrated to effectively treat the P. aeruginosa infection and accelerate the healing process at the wound site of mouse. Owing to the light irradiation triggered targeted imaging and enhanced bactericidal capacities, P2pCe6 hold great potential to serve as a potent PS for mediating the phototheranostics of gram-negative bacterial infection.

All-hydrocarbon stapling enables improvement of antimicrobial activity and proteolytic stability of peptide Figainin 2.

Figainin 2 is a cationic, hydrophobic, α-helical host-defense peptide with 28 residues, which was isolated from the skin secretions of the Chaco tree frog. It shows potent inhibitory activity against both Gram-negative and Gram-positive pathogens and has garnered considerable interest in developing novel classes of natural antibacterial agents. However, as a linear peptide, conformational flexibility and poor proteolytic stability hindered its development as antibacterial agent. To alleviate its susceptibility to proteolytic degradation and improve its antibacterial activity, a series of hydrocarbon-stable analogs of Figainin 2 were synthesized and evaluated for their secondary structure, protease stability, antimicrobial, and hemolytic activities. Among them, F2-12 showed significant improvement in protease resistance and antimicrobial activity compared to that of the template peptide. This study provides a promising strategy for the development of antimicrobial drugs.

Design, Synthesis, and Anti-Osteoporotic Characterization of Arginine N-Glycosylated Teriparatide Analogs via the Silver-catalyzed Solid-Phase Glycosylation Strategy.

In spite of effective antiosteoporosis potency, teriparatide, a bone-building agent approved by the FDA (Food and Drug Administration), was proven to exhibit various side effects. In our previous work, we developed a universal strategy for synthesizing arginine N-glycosylated peptides termed silver-promoted solid-phase glycosylation (SSG) strategy. However, it is unknown whether the SSG strategy can be applied in the peptide drug design. Herein, we first reported the optimization of teriparatide via SSG strategy. Using Arg20 and/or Arg25 as the modifying positions, three series of arginine N-glycosylated teriparatide analogs were successfully synthesized, of which the introduced sugar groups included glucose, galactose, mannose, rhamnose, ribose, 2-acetamino-2-deoxy-glucose, xylose, lactose, and maltose. Among the 27 arginine N-glycosylated derivatives, Arg20-xylose and Arg25-maltose teriparatide analogs, termed PTH-1g and PTH-2i, respectively, indicated enhanced serum stability and significantly improved antiosteoporotic activities in vitro and in vivo compared with the native counterpart. They may serve as effective therapeutic candidates for treating osteoporosis.

Discovery of a novel antifungal agent: All-hydrocarbon stapling modification of peptide Aurein1.2.

Aurein1.2 is secreted by the Australian tree frog Litoria aurea and is active against a broad range of infectious microbes including bacteria, fungi, and viruses. Its antifungal potency has garnered considerable interest in developing novel classes of natural antifungal agents to fight pathogenic infection by fungi. However, serious pharmacological hurdles remain, hindering its clinical translation. To alleviate its susceptibility to proteolytic degradation and improve its antifungal activity, six conformationally locked peptides were synthesized through hydrocarbon stapling modification and evaluated for their physicochemical and antifungal parameters. Among them, SAU2-4 exhibited significant improvement in helicity levels, protease resistance, and antifungal activity compared to the template linear peptide Aurein1.2. These results confirmed the prominent role of hydrocarbon stapling modification in the manipulation of peptide pharmacological properties and enhanced the application potential of Aurein1.2 in the field of antifungal agent development.

Enhanced Tumor Targeting and Penetration of Proteolysis-Targeting Chimeras through iRGD Peptide Conjugation: A Strategy for Precise Protein Degradation in Breast Cancer.

Proteolysis-targeting chimeras (PROTACs) have recently emerged as a promising technology for drug development. However, poor water solubility, limited tissue selectivity, and inadequate tumor penetration pose significant challenges for PROTAC-based therapies in cancer treatment. Herein, we developed an iRGD-PROTAC conjugation strategy utilizing tumor-penetrating cyclic peptide iRGD (CRGDK/RGPD/EC) to deliver PROTACs deep into breast cancer tissues. As a conceptual validation study, iRGD peptides were conjugated with a bromodomain-containing protein 4 (BRD4) PROTAC through a GSH-responsive linker. The resulting iRGD-PROTAC conjugate iPR showed enhanced water solubility, tumor-targeting capability, and penetration within tumor tissues, resulting in increased antibreast cancer efficacy in animal models and patient-derived organoids. This study demonstrates the advantages of combining iRGD and PROTACs in improving drug delivery and highlights the importance of tissue selectivity and penetration ability in PROTAC-based therapeutics.

Corrigendum: Design, synthesis, and antitumor activity study of all-hydrocarbon-stapled B1-Leu peptides.

[This corrects the article DOI: 10.3389/fchem.2022.840131.].

Benzyl stapled modification and anticancer activity of antimicrobial peptide A4K14-Citropin 1.1.

A4K14-Citropin 1.1 (GLFAVIKKVASVIKGL-NH2) is a derived antimicrobial peptide (AMP) with a more stable α-helical structure at the C-terminal compared to prototype Citropin 1.1 which was obtained from glandular skin secretions of Australian freetail lizards. In a previous report, A4K14-Citropin 1.1 has been considered as an anti-cancer lead compound. However, linear peptides are difficult to maintain stable secondary structure, resulted in poor pharmacokinetic properties. In this study, we designed and synthesized a series of benzyl-stapled derivatives of A4K14-Citropin 1.1. And their physical and chemical properties, as well as biological activity, were both explored. The result showed that AC-CCSP-2-o and AC-CCSP-3-o exhibited a higher degree of helicity and greater anti-cancer activity compared with the prototype peptide. Besides, there was no significant difference in the hemolytic effect between the stapled peptides and the prototype peptide. AC-CCSP-2-o and AC-CCSP-3-o could serve as promising anti-cancer lead compounds for the novel anti-cancer drug development.

Cyclobutane-bearing restricted anchoring residues enabled geometry-specific hydrocarbon peptide stapling.

Stapled peptides are regarded as the promising next-generation therapeutics because of their improved secondary structure, membrane permeability and metabolic stability as compared with the prototype linear peptides. Usually, stapled peptides are obtained by a hydrocarbon stapling technique, anchoring from paired olefin-terminated unnatural amino acids and the consequent ring-closing metathesis (RCM). To investigate the adaptability of the rigid cyclobutane structure in RCM and expand the chemical diversity of hydrocarbon peptide stapling, we herein described the rational design and efficient synthesis of cyclobutane-based conformationally constrained amino acids, termed (E)-1-amino-3-(but-3-en-1-yl)cyclobutane-1-carboxylic acid (E7) and (Z)-1-amino-3-(but-3-en-1-yl)cyclobutane-1-carboxylic acid (Z7). All four combinations including E7-E7, E7-Z7, Z7-Z7 and Z7-E7 were proven to be applicable in RCM-mediated peptide stapling to afford the corresponding geometry-specific stapled peptides. With the aid of the combined quantum and molecular mechanics, the E7-E7 combination was proven to be optimal in both the RCM reaction and helical stabilization. With the spike protein of SARS-CoV-2 as the target, a series of cyclobutane-bearing stapled peptides were obtained. Among them, E7-E7 geometry-specific stapled peptides indeed exhibit higher α-helicity and thus stronger biological activity than canonical hydrocarbon stapled peptides. We believe that this methodology possesses great potential to expand the scope of the existing peptide stapling strategy. These cyclobutane-bearing restricted anchoring residues served as effective supplements for the existing olefin-terminated unnatural amino acids and the resultant geometry-specific hydrocarbon peptide stapling provided more potential for peptide therapeutics.

Discovery of an orally effective double-stapled peptide for reducing ovariectomy-induced bone loss in mice.

Stapled peptides with significantly enhanced pharmacological profiles have emerged as promising therapeutic molecules due to their remarkable resistance to proteolysis and performance to penetrate cells. The all-hydrocarbon peptide stapling technique has already widely adopted with great success, yielding numerous potent peptide-based molecules. Based on our prior efforts, we conceived and prepared a double-stapled peptide in this study, termed FRNC-1, which effectively attenuated the bone resorption capacity of mature osteoclasts in vitro through specific inhibition of phosphorylated GSK-3ß. The double-stapled peptide FRNC-1 displayed notably improved helical contents and resistance to proteolysis than its linear form. Additionally, FRNC-1 effectively prevented osteoclast activation and improved bone density for ovariectomized (OVX) mice after intravenous injection and importantly, after oral (intragastric) administration. The double-stapled peptide FRNC-1 is the first orally effective peptide that has been validated to date as a therapeutic candidate for postmenopausal osteoporosis (PMOP).

Unleashing the potential of natural biological peptide Macropin: Hydrocarbon stapling for effective breast cancer treatment.

The identification of novel candidate molecules with the potential to revolutionize the treatment of breast cancer holds profound clinical significance. Macropin (Mac)-1, derived from the venom of wild bees, emerges as an auspicious therapeutic agent for combating breast cancers. Nevertheless, linear peptides have long grappled with the challenges of traversing cell membranes and succumbing to protease hydrolysis. To address this challenge, the present study employed hydrocarbon stapling modification to synthesize a range of stapled Mac-1 peptides, which were comprehensively evaluated for their chemical and biological properties. Significantly, Mac-1-sp4 exhibited a remarkable set of improvements, including enhanced helicity, proteolytic stability, cell membrane permeability, induction of cell apoptosis, in vivo antitumor activity, and inhibition of tubulin polymerization. This study explores the significant impact of the hydrocarbon stapling technique on the secondary structure, hydrolase stability, and biological activity of Mac-1, shedding light on its potential as a revolutionary and potent anti-breast cancer therapy. The findings establish a strong basis for the development of innovative and highly effective anti-tumor treatments.

The recent progress of peptide regulators for the Wnt/ß-catenin signaling pathway.

Wnt signaling plays an important role in many biological processes such as stem cell self-renewal, cell proliferation, migration, and differentiation. The ß-catenin-dependent signaling pathway mainly regulates cell proliferation, differentiation, and migration. In the Wnt/ß-catenin signaling pathway, the Wnt family ligands transduce signals through LRP5/6 and Frizzled receptors to the Wnt/ß-catenin signaling cascades. Wnt-targeted therapy has garnered extensive attention. The most commonly used approach in targeted therapy is small-molecule regulators. However, it is difficult for small-molecule regulators to make great progress due to their inherent defects. Therapeutic peptide regulators targeting the Wnt signaling pathway have become an alternative therapy, promising to fill the gaps in the clinical application of small-molecule regulators. In this review, we describe recent advances in peptide regulators for Wnt/ß-catenin signaling.

Design, synthesis and antitumor activity of Ascaphin-8 derived stapled peptides based on halogen-sulfhydryl click chemical reactions.

Ascaphin-8 (GFKDLLKGAAKALVKTVLF-NH2), isolated from the norepinephrine-stimulated skin secretion of the North American-tailed frog Ascaphus truei, is a C-terminal α-helical antimicrobial peptide with potential antitumor activity. However, linear peptides are difficult to be applied directly as drugs because of their inherent defects, such as low hydrolytic enzyme tolerance and poor structural stability. In this study, we designed and synthesized a series of stapled peptides based on Ascaphin-8 via thiol-halogen click chemistry. Most of the stapled peptide derivatives showed enhanced antitumor activity. Among them, A8-2-o and A8-4-Dp had the most improved structural stability, stronger hydrolytic enzyme tolerance and highest biological activity. This research may provide a reference for the stapled modification of other similar natural antimicrobial peptides.

pH-Activated Ce-Doped Molybdenum Oxide Nanoclusters for Tumor Microenvironment Responsive Photothermal and Chemodynamic Therapy.

Molybdenum-based nanomaterials have shown promise for anticancer treatment due to their strong photothermal and redox-activated capabilities. Herein, we have fabricated cerium-doped MoOx (Ce-MoOv) with tunable Mo/Ce molar ratios by a one-pot method and investigated their effect on chemodynamic therapy (CDT) and photothermal therapy (PTT). It is found that Ce-MoOv can self-assemble into nanoclusters in acidic conditions and the increasing Ce amount will generate oxygen vacancy defects and induce the valence change of Mo6+/Mo5+ and Ce4+/Ce3+, which leads to strong near-infrared absorption with high photothermal conversion efficiency of 71.31 and 49.86% for 808 and 1064 nm. Other than photothermal conversion, the materials demonstrate pH-/glutathione (GSH)-activated photoacoustic (PA) imaging capability in vitro. In addition, Ce-MoOv acts as a CDT reagent capable of converting endogenous H2O2 to two types of reactive oxygen species (•OH, 1O2) while depleting GSH. Ce-MoOv demonstrates an excellent therapeutic effect against HCT116 cells and effectively reduces the intracellular GSH level and significantly increases the number of reactive radicals under 1064 nm laser irradiation as compared with the no-laser group in vitro. This work provides a new paradigm using lanthanide-doped polymetallic oxides for pH-/GSH-responsive photothermal/chemodynamic therapy with PA imaging ability.

The regulatory metabolic networks of the Brassica campestris L. hairy roots in response to cadmium stress revealed from proteome studies combined with a transcriptome analysis.

Brassica campestris L., a cadmium (Cd) hyperaccumulating herbaceous plant, is considered as a promising candidate for the bioremediation of Cd pollution. However, the molecular mechanisms regulating these processes remain unclear. The present work, using proteome studies combined with a transcriptome analysis, was carried out to reveal the response mechanisms of the hairy roots of Brassica campestris L. under Cd stress. Significant tissue necrosis and cellular damage occurred, and Cd accumulation was observed in the cell walls and vacuoles of the hairy roots. Through quantitative proteomic profiling, a total of 1424 differentially expressed proteins (DEPs) were identified, and are known to be enriched in processes including phenylalanine metabolism, plant hormone signal transduction, cysteine and methionine metabolism, protein export, isoquinoline alkaloid biosynthesis and flavone biosynthesis. Further studies combined with a transcriptome analysis found that 118 differentially expressed genes (DEGs) and their corresponding proteins were simultaneously up- or downregulated. Further Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analysis of the 118 shared DEGs and DEPs indicated their involvement in calcium, ROS and hormone signaling-mediated response, including regulation of carbohydrate and energy metabolism, biosynthesis of GSH, PCs and phenylpropanoid compounds that play vital roles in the Cd tolerance of Brassica campestris L. Our findings contribute to a better understanding of the regulatory networks of Brassica campestris L. under Cd stress, as well as provide valuable information on candidate genes (e.g., BrPAL, BrTAT, Br4CL, BrCDPK, BrRBOH, BrCALM, BrABCG1/2, BrVIP, BrGCLC, BrilvE, BrGST12/13/25). These results are of particular importance to the subsequent development of promising transgenic plants that will hyperaccumulate heavy metals and efficient phytoremediation processes.

Methionine restriction promotes cGAS activation and chromatin untethering through demethylation to enhance antitumor immunity.

Cyclic GMP-AMP synthase (cGAS) is the major sensor for cytosolic DNA and activates type I interferon signaling and plays an essential role in antitumor immunity. However, it remains unclear whether the cGAS-mediated antitumor activity is affected by nutrient status. Here, our study reports that methionine deprivation enhances cGAS activity by blocking its methylation, which is catalyzed by methyltransferase SUV39H1. We further show that methylation enhances the chromatin sequestration of cGAS in a UHRF1-dependent manner. Blocking cGAS methylation enhances cGAS-mediated antitumor immunity and suppresses colorectal tumorigenesis. Clinically, cGAS methylation in human cancers correlates with poor prognosis. Thus, our results indicate that nutrient stress promotes cGAS activation via reversible methylation, and suggest a potential therapeutic strategy for targeting cGAS methylation in cancer treatment.

Establishment of hairy root system of transgenic IRT1 brassica campestris L. and preliminary study of its effect on cadmium enrichment.

Cadmium (Cd) is the main heavy metal pollutant in soil. The combination of genetic engineering technology and Rizobium rhizogenes mediated technology can effectively improve the enrichment efficiency of heavy metals in super accumulators and reduce soil heavy metal pollution. In this study, the transgenic hairy root system containing the IRT1 gene of Cd hyperaccumulator-Brassica campestris L. was successfully constructed by the R. rhizogenes mediated method (IRT1 gene come from Arabidopsis thaliana). The hairy roots of each subculture can grow stably within 6 weeks, and IRT1 gene will not be lost within 50 subcultures., which is detected using PCR method. The results of Cd enrichment experiments showed that after treatment with 100 µmol/L Cd for 14 days, the growth state of transgenic IRT1 hairy roots only showed slight browning. Also, the accumulation value of Cd reached 331.61 µg/g and the enrichment efficiency of transgenic IRT1 hairy roots was 13.8% higher than that of wild-type hairy roots. Western blotting results showed that the expression of IRT1 protein in transgenic hairy roots was significantly higher than that of wild-type hairy roots under Cd stress. The above results indicated that the overexpression of IRT1 gene can help B. campestris L. hairy roots to effectively cope with Cd stress and improve its ability to enrich Cd.

In this study, the transgenic hairy root system containing the IRT1 gene of Cd hyperaccumulator-Brassica campestris L. was successfully constructed by the Rizobium rhizogenes mediated method. At the same time, the growth state and cadmium enrichment efficiency of transgenic hairy roots under different concentrations of Cd stress were studied. Overexpression of IRT1 gene can effectively improve the tolerance of hairy root to Cd. The enrichment efficiency of transgenic IRT1 hairy roots was 13.8% higher than that of wild-type hairy roots. The transgenic IRT1 hairy root system established in this study can be used as a reliable experimental model for the study of Cd adsorption mechanism, and can be further regenerated to obtain transgenic IRT1 B. campestris L. plants for the study of heavy metal Cd pollution remediation.

DUCNP@Mn-MOF/FOE as a Highly Selective and Bioavailable Drug Delivery System for Synergistic Combination Cancer Therapy.

Heterostructures comprising lanthanide-doped upconversion nanoparticles (DUCNPs) and metal-organic frameworks (MOFs) are emerging as promising nanosystems for integrating medical diagnosis and treatment. Here, the DUCNP@Mn-MOF nanocarrier was developed, which showed good efficiency for loading and delivering a cytotoxic antitumor agent (3-F-10-OH-evodiamine, FOE). The combined advantages of the pH-responsive and peroxidase-like properties of Mn-MOF and the unique optical features of DUCNPs granted the DUCNP@Mn-MOF/FOE system synergistic chemodynamic and chemotherapeutic effects. The DUCNP@Mn-MOF nanocarrier effectively overcame the intrinsic limitations of FOE, such as its unfavorable physicochemical properties and limited in vivo potency. This complexed nanosystem was responsive to the tumor microenvironment and showed excellent tumor targeting capability. Thus, DUCNP@Mn-MOF/FOE exhibited highly selective and bioavailable drug delivery properties and is promising for cancer therapy. In a mouse breast cancer model, DUCNP@Mn-MOF/FOE inhibited tumor growth without significant toxicity. Therefore, the proposed nanosystem represents a promising theragnostic platform for multimodal combination diagnosis and therapy of tumors.