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.

Enhancing anticancer treatment: Development of cRGD-Conjugated F-OH-Evo prodrugs for targeted delivery.

Small molecule drugs sourced from natural products are pivotal for novel therapeutic discoveries. However, their clinical deployment is often impeded by non-specific activity and severe adverse effects. This study focused on 3-fluoro-10-hydroxy-Evodiamine (F-OH-Evo), a potent derivative of Evodiamine, whose development is curtailed due to suboptimal tumor selectivity and heightened cytotoxicity. By harnessing the remarkable stability, specificity, and αvß3 integrin affinity of c(RGDFK), a novel prodrug by conjugating F-OH-Evo with cRGD was synthesized. This innovative prodrug substantially enhanced the tumor-specific targeting of F-OH-Evo and improved the anti-tumor activities. Among them, compound 3c demonstrated the best selective inhibitory activity toward U87 cancer cells in vitro. It selectively enterd U87 cells by binding to αvß3 integrin, releasing the parent molecule under the dual response of ROS and GSH to exert inhibitory activity on topo I. The results highlight the potential of cRGD-conjugated prodrugs in targeted cancer therapy. This approach signifies a significant advancement in developing safer and more effective chemotherapy drugs, emphasizing the role of prodrug strategies in overcoming the limitations of traditional cancer treatments.

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.

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.

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.

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.

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.

Configuration-Specific Antibody for Bacterial Heptosylation: An Antiadhesion Therapeutic Strategy.

Alternative antibacterial therapies refractory to existing mechanisms of antibiotic resistance are urgently needed. One such attractive therapy is to inhibit bacterial adhesion and colonization. Ser O-heptosylation (Ser O-Hep) on autotransporters of Gram-negative bacteria is a novel glycosylation and has been proven to be essential for bacterial colonization. Herein, we chemically synthesized glycopeptides containing this atypical glycan structure and an absolute C6 configuration through the assembly of Ser O-Hep building blocks. Using glycopeptides as haptens, we generated first-in-class poly- and monoclonal antibodies, termed Anti-SerHep1a and Anti-SerHep1b, that stereoselectively recognize Ser O-heptosylation (d/l-glycero) with high specificity in vitro and in vivo. Importantly, these antibodies effectively blocked diffusely adhering Escherichia coli 2787 adhesion to HeLa cells and in mice in a dose- and Ser O-Hep-dependent manner. Together, these antibodies represent not only useful tools for the discovery of unknown serine O-heptosylated proteins bearing various C6 chiral centers but also a novel class of antiadhesion therapeutic agents for the treatment of bacterial infection.

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.

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.

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.

A toolbox of diverse arginine N-glycosylated peptides and specific antibodies.

Among posttranslational modifications, atypical arginine N-glycosylation has drawn increasing interest due to its fundamental role in various cellular procedures and signaling pathways. The efficient synthesis of arginine N-glycosylated substrates, as well as the generation of specific antibodies, remains challenging. This work describes the efficient synthesis of diverse arginine N-glycosylated peptides, in a process termed silver-promoted solid-phase glycosylation (SGG). There are two key features of the SSG strategy: (i) robust synthesis of gram-scale S-alkyl-isothiourea glycosyl donors facilitates the subsequent SSG procedure and (ii) the simultaneous introduction of both the side-chain sugar motif and arginine residue. Notably, our findings, combined with our previous results, provide a toolbox containing diverse S-alkyl-isothiourea glycosyl donors (glucose, galactose, mannose, ribose, xylose, lactose and maltose) as well as the corresponding Arg N-glycosylated peptides. In addition, our toolbox is shown to help investigate specific antibodies and identify multiple potent and precise biochemical tools for exploring arginine N-glycosylation.

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.

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.

Visible-Light-Induced Difunctionalization of Alkenyl Ketones with α-Carbonyl Alkyl Bromide: Concomitant Installation of C-C Bonds.

The visible-light-promoted difunctionalization of alkenyl ketones has been developed for easy access of various tetralones, cyclopropane, or alkenyl migration compounds. With fac-[Ir(ppy)3] as the photocatalyst, alkenyl ketones captured the α-carbonyl alkyl radical and evolved through intramolecular cyclization and the elimination of a proton to give the difunctionalized products. This strategy is characterized by good yields, mild reaction conditions, and outstanding functional group tolerance.

Stapled Helical Peptides Bearing Different Anchoring Residues.

A large proportion of protein-protein interactions (PPIs) occur between a short peptide and a globular protein domain; the peptides involved in surface interactions play important roles, and there is great promise for using peptide motifs to interfere with protein interactions. Peptide inhibitors show more promise in blocking large surface protein interactions compared to small molecule inhibitors. However, peptides have drawbacks including poor stability against circulating proteolytic enzymes and an intrinsic inability to penetrate cell membranes. Stapled helical peptides, by adopting a preformed, stable α-helical conformation, exhibit improved proteolytic stability and membrane permeability compared to linear bioactive peptides. In this review, we summarize the broad aspects of peptide stapling for chemistry, biophysics, and biological applications and specifically highlight the methodology by providing an inventory of different anchoring residues categorized into two natural amino acids, two nonnatural amino acids, or a combination of natural and nonnatural amino acids. Additional advantages of specific peptide stapling techniques, including but not limited to reversibility, bio-orthogonal reactivity, and photoisomerization, are also discussed individually. This review is expected to provide a broad reference for the rational design of druggable stapled peptides targeting therapeutic proteins, particularly those involved in PPIs, by considering the impact of anchoring residues, functional cross-linkers, physical staple length, staple components, and the staple motif on the biophysical properties of the peptides.

Hydrocarbon stapling modification of peptide alyteserin-2a: Discovery of novel stapled peptide antitumor agents.

Alyteserin-2a (ILGKLLSTAAGLLSNLNH2 ) is isolated from the skin exudates of midwife toad and has a wide range of biological applications. However, the use of alyteserin-2a as an antitumor agent is limited due to its structural flexibility. In this study, a series of stapled peptides were prepared through hydrocarbon stapling modification without destroying the key residues, and their chemical and biological properties were further evaluated for enhancing the application potential of alyteserin-2a in the field of antitumor drugs development. Among them, alyteserin-2a-Sp3 displayed significant improvement in helicity levels, protease resistance, and antitumor activity compared to that of the template peptide alyteserin-2a, indicating that alyteserin-2a-Sp3 had a potential to become a lead compound for the development of novel antitumor drugs. This study confirms the important effect of hydrocarbon stapling strategy on the secondary structure, hydrolase stability, and biological activity of alyteserin-2a.

Design, synthesis, and anti-tumor activities of novel Brevinin-1BYa peptidomimetics.

Brevinin-1BYa is an amphibian skin-derived peptide that exhibits promising anti-microbial activity against gram-positive and -negative bacteria. However, the anti-tumor activity of Brevinin-1BYa remains unclear, and, more importantly, its therapeutic application is limited owing to its poor protease and reduction stability. In this study, a series of novel Brevinin-1BYa derivatives, including O-linked N-acetyl-glucosamine glyclopeptides and disulfide bond mimetics, were designed and synthesized. Additionally, their anti-tumor activity against human prostate cancer cell line C4-2B, human NSCLC cell line A549 (adenocarcinoma), and human hepatoma cells line HuH-7 was investigated. Among these, the thioether bridge substituted peptidomimetic Brevinin-1BYa-3 displayed improved reduction stability, more stable secondary structure, greater protease stability, and increased anti-tumor activity compared with the original peptide, rendering it a promising leading compound for drug development, particularly for applications against malignant tumors.

Design, synthesis, and biological evaluation of stapled ascaphin-8 peptides.

Ascaphin-8 is an α-helical anti-tumor and antimicrobial peptide containing 19 residues, which was isolated from norepinephrine-stimulated skin secretions of the North American tailed frog Ascaphus truei. To improve both its stability and biological activities, a series of hydrocarbon-stapled analogs of Ascaphin-8 were synthesized and investigated for their potential antiproliferative activities. The activity studies were evaluated using the CCK-8 method and colony formation assay on human cancer cell lines. Ascaphin-8-3, as the most active peptide, showed a stronger inhibition effect when compared with the parent peptide for the tested cell lines. In addition, the effect of Ascaphin-8-3 on inhibiting the metastatic capabilities of A549 cells was more powerful than that of the parent peptide. This peptide derivative showed potentiality for further optimization in antitumor drugs.

Constructions of Beyond-Birthday Secure PRFs from Random Permutations, Revisited.

In CRYPTO 2019, Chen et al. showed how to construct pseudorandom functions (PRFs) from random permutations (RPs), and they gave one beyond-birthday secure construction from sum of Even-Mansour, namely SoEM22 in the single-key setting. In this paper, we improve their work by proving the multi-key security of SoEM22, and further tweaking SoEM22 but still preserving beyond birthday bound (BBB) security. Furthermore, we use only one random permutation to construct parallelizable and succinct beyond-birthday secure PRFs in the multi-key setting, and then tweak this new construction. Moreover, with a slight modification of our constructions of tweakable PRFs, two parallelizable nonce based MACs for variable length messages are obtained.