Dual-functional Peptide Research Articles

Presenting dual-functional peptides on implant surface to direct in vitro osteogenesis and in vivo osteointegration

The complex biological process of osseointegration and the bio-inertness of bone implants are the major reasons for the high failure rate of long-term implants, and have also promoted the rapid development of multifunctional implant coatings in recent years. Herein, through the special design of peptides, we use layer-by-layer assembly technology to simultaneously display two peptides with different biological functions on the implant surface to address this issue. A variety of surface characterization techniques (ellipsometry, atomic force microscopy, photoelectron spectroscopy, dissipation-quartz crystal microbalance) were used to study in detail the preparation process of the dual peptide functional coating and the physical and chemical properties, such as the composition, mechanical modulus, stability, and roughness of the coating. Compared with single peptide functional coatings, dual-peptide functionalized coatings had much better performances on antioxidant, cellular adhesion in early stage, proliferation and osteogenic differentiation in long term, as well as in vivo osteogenesis and osseointegration capabilities. These findings will promote the development of multifunctional designs in bone implant coatings, as a coping strategy for the complexity of biological process during osteointegration.

A blood–brain barrier- and blood–brain tumor barrier-penetrating siRNA delivery system targeting gliomas for brain tumor immunotherapy

Glioma is recognized as the most infiltrative and lethal form of central nervous system tumors and is known for its limited response to standard therapeutic interventions, high recurrence rate, and unfavorable prognosis. Recent progress in gene and immunotherapy presents a renewed sense of optimism in the treatment of glioblastoma. However, the barriers to overcome include the blood–brain barrier (BBB) and the blood–brain tumor barrier (BBTB), as well as the suppressive immune microenvironment. Overcoming these barriers remains a significant challenge. Here, we developed a lipid nanoparticle platform incorporating a dual-functional peptide (cholesterol-DP7-ACP-T7-modified DOTAP or DAT-LNP) capable of targeting glioma across the BBB and BBTB for brain tumor immunotherapy. This system was designed to achieve two key functions. First, the system could effectively penetrate the BBB during accumulation within brain tissue following intravenous administration. Second, this system enhances the maturation of dendritic cells, the polarization of M1 macrophages, and the activation of cytotoxic CD8+ T cells. This multifaceted approach effectively mitigates the immunosuppressive tumor microenvironment of glioma and promotes robust antitumor immune responses. Overall, the intravenous administration of the delivery system designed in this study demonstrates significant therapeutic potential for glioma and holds promising applications in the field of cancer immunotherapy.

Purification, identification and molecular docking of dual-functional peptides derived from corn protein hydrolysates with antioxidant and antiadhesive activity against Helicobacter pylori.

More than 50% of the world's population is infected with Helicobacter pylori, which is classified as a group I carcinogen by the World Health Organization (WHO). Corn protein dual-functional peptides were identified and functionally analyzed in vitro and in silico. Two novel dual-functional peptides were identified as Cys-Gln-Asp-Val-Pro-Leu-Leu (CQDVPLL, CQ7) and Thr-Ile-Phe-Pro-Gln-Cys (TIFPQC, TI6) using nanoscale liquid chromatography coupled to tandem mass spectrometry (nano-LC-MS/MS). The antiadhesive effects against H. pylori of CQ7 and TI6 were 45.17 ± 2.41% and 48.62 ± 1.84% at 4 mg mL-1 , respectively. In silico prediction showed that CQ7 and TI6 had good physicochemical properties. Molecular docking demonstrated that CQ7 and TI6 could bind to the adhesins BabA and SabA by hydrophobic interactions and hydrogen bonds, preventing H. pylori infection. Moreover, CQ7 showed strong antioxidant activity due to its unique amino acid composition. The present study demonstrated that the identified peptides, CQ7 and TI6, possess antioxidant and antiadhesive effects, preventing H. pylori infection and alleviating oxidative injury to the gastric mucosa. © 2023 Society of Chemical Industry.

Peptide and peptidomimetic tyrosinase inhibitors.

Melanin, which is produced by melanocytes and spread over keratinocytes, is responsible for human skin browning. There are several processes involved in melanogenesis, mostly prompted by enzymatic activities. Tyrosinase (TYR), a copper containing metalloenzyme, is considered the main actor in melanin production, as it catalyzes two crucial steps that modify tyrosine residues in dopaquinone. For this reason, TYR inhibition has been exploited as a possible mechanism of modulation of hyper melanogenesis. There are various types of molecules used to block TYR activity, principally used as skin whitening agents in cosmetic products, e.g., tretinoin, hydroquinone, azelaic acid, kojic acid, arbutin and peptides. Peptides are highly valued for their versatile nature, making them promising candidates for various functions. Their specificity often leads to excellent safety, tolerability, and efficacy in humans, which can be considered their primary advantage over traditional small molecules. There are several examples of tyrosinase inhibitor peptides (TIPs) operating as possible hypo-pigmenting agents, which can be classified according to their origin: natural, hybrid or synthetically produced. Moreover, the possibility of variating their backbones, introducing non-canonical amino acids or modifying one or more peptide bond(s), to obtain peptidomimetic molecules, is an added value to avoid or delay proteolytic activity, while the possibility of conjugation with other bioactive peptides or organic moieties can bring other specific activity leading to dual-functional peptides.

Discovery and Characterization of a Dual-Function Peptide Derived from Bitter Gourd Seed Protein Using Two Orthogonal Bioassay-Guided Fractionations Coupled with In Silico Analysis.

The hydrolysate of bitter gourd seed protein, digested by the combined gastrointestinal proteases (BGSP-GPs), exhibited the most potent inhibition on angiotensin-I-converting enzyme (ACE) with an IC50 value of 48.1 ± 2.0 µg/mL. Using two independent bioassay-guided fractionations, fraction F5 from reversed-phase chromatography and fraction S1 from strong cation exchange chromatography exhibited the highest ACE inhibitory (ACEI) activity. Three identical peptides were simultaneously detected from both fractions and, based on the in silico appraisal, APLVSW (AW6) was predicted as a promising ACEI peptide. Their dipeptidyl peptidase-IV (DPP4) inhibitory (DPP4I) activity was also explored. The IC50 values of AW6 against ACE and DPP4 were calculated to be 9.6 ± 0.3 and 145.4 ± 4.4 µM, respectively. The inhibitory kinetics and intermolecular interaction studies suggested that AW6 is an ACE competitive inhibitor and a DPP4 non-competitive inhibitor. The quantities of AW6 in BGSP-GP hydrolysate, fractions F5 and S1, were also analyzed using liquid chromatography-tandem mass spectrometry. Notably, AW6 could resist hydrolysis in the human gastrointestinal tract according to the result of the simulated gastrointestinal digestion. To the best of our knowledge, this is the first discovery and characterization of a dual-function (ACEI and DPP4I activities) peptide derived from bitter gourd seed protein.

Characterization and Classification In Silico of Peptides with Dual Activity (Antimicrobial and Wound Healing).

The growing challenge of chronic wounds and antibiotic resistance has spotlighted the potential of dual-function peptides (antimicrobial and wound healing) as novel therapeutic strategies. The investigation aimed to characterize and correlate in silico the physicochemical attributes of these peptides with their biological activity. We sourced a dataset of 207 such peptides from various peptide databases, followed by a detailed analysis of their physicochemical properties using bioinformatic tools. Utilizing statistical tools like clustering, correlation, and principal component analysis (PCA), patterns and relationships were discerned among these properties. Furthermore, we analyzed the peptides' functional domains for insights into their potential mechanisms of action. Our findings spotlight peptides in Cluster 2 as efficacious in wound healing, whereas Cluster 1 peptides exhibited pronounced antimicrobial potential. In our study, we identified specific amino acid patterns and peptide families associated with their biological activities, such as the cecropin antimicrobial domain. Additionally, we found the presence of polar amino acids like arginine, cysteine, and lysine, as well as apolar amino acids like glycine, isoleucine, and leucine. These characteristics are crucial for interactions with bacterial membranes and receptors involved in migration, proliferation, angiogenesis, and immunomodulation. While this study provides a groundwork for therapeutic development, translating these findings into practical applications necessitates additional experimental and clinical research.

Health-promoting activities of Moringa oleifera Lam. seeds protein hydrolysates and its ultra-filtered peptide fractions

Moringa oleifera (MO) seeds treated as a new resource food rich in protein (37.48%), but, little know the bioactive benefits and peptide composition of protein hydrolysates. In this study, ultrafiltrated peptide fractions of MO seed proteins hydrolysates (MOSPH) were analyzed for their antioxidant, anti-hypertension and sleep-promoting activities. LC-MS/MS was used to identify the peptide composition of the molecular weight (MW) <3 kDa fraction. The results showed that MOSPH and the lower MW peptides fraction, especially the MW <3 kDa fraction, exhibited higher free radical scavenging and reducing capacity, in vitro angiotensin I-converting enzyme (ACE) inhibitory capacity and vasodilatory and anti-hypertensive effects on spontaneously hypertensive rats (SHR) after the development of hypertension, sleep-promoting and anticonvulsant effects in male ICR mice. Furthermore, four potential bioactive peptides (pepsite score > 0.3) from MW <3 kDa fraction of MOSPH were explored via in silico approach, of which a novel dual-function peptide IWHHTFYNELR (IR-11) exhibited higher ACE inhibitory activity (IC50 = 1.057 mg/mL) and DPPH•-clearing activity (IC50 = 0.404 mg/mL). Molecular docking indicates that IR-11 interacted with ACE and DPPH• through hydrogen bonding and hydrophobic forces. Our results suggested that MO seed proteins peptides can be a potent functional agents for developing new functional food.

Cell and Material Specific Dual-Functional Peptides for Enhancing Mesenchymal Stem Cell Migration for Bone Tissue Engineering

Cell and Material Specific Dual-Functional Peptides for Enhancing Mesenchymal Stem Cell Migration for Bone Tissue Engineering

An engineered dual-functional peptide with high affinity to demineralized dentin enhanced remineralization efficacy in vitro and in vivo.

Dental caries continues to be a major global public health problem. Remineralization of demineralized dentin is regarded as one of the hotspots in the current study in the treatment of dental caries. However, traditional remineralization agents, which usually lack the ability to bind to demineralized dentin collagen, are easily removed by the fluids in the oral cavity, thus decreasing the remineralization efficacy. Non-collagenous proteins (NCPs) have significant effects on the biomineralization of dentin due to their dual high binding capacity to the collagen fibers and minerals. But NCPs are hard to extract, store and use directly. Inspired by the biological behavior of NCPs, in this study, we selected two functional sequences of NCPs to develop a novel and engineered dual-functional peptide (which is referred to as CYP) with collagen-binding and mineral-absorbing capability. The binding ability of CYP to collagen fibers and demineralized dentin was investigated, and the results suggested that CYP was endowed with good binding capacity to demineralized dentin, which could resist the washing of the fluid. In addition, we confirmed that CYP exerted formidable remineralization effects in collagen fibers and demineralized dentin following an in vitro remineralization regimen. Furthermore, the dual functions of CYP with good biocompatibility can simultaneously bind collagen and induce nanocrystal precipitation, thereby significantly absorbing calcium and phosphorus ions to form regenerated minerals for reversing the tooth decay process in the rat caries model. Overall, the dual functional peptide CYP fabricated in this study provides an ideal and smart strategy for dentin remineralization and the treatment of caries.

An engineered dual function peptide to repair fractured bones.

Targeted drug delivery, often referred to as "smart" drug delivery, is a process whereby a therapeutic drug is delivered to specific parts of the body in a manner that increases its concentration at the desired sites relative to others. This approach is poised to revolutionize medicine as exemplified by the recent FDA approval of Cytalux (FDA approves pioneering drug for ovarian cancer surgery - Purdue University News) which is a folate-receptor targeted intraoperative near infrared (NIR) imaging agent that was developed in our laboratories. Fracture-associated morbidities and mortality affect a significant portion of world population. United states, Canada and Europe alone spent $48 billion in treating osteoporosis related fractures although this number doesn't count the economic burden due to loss in productivity. It is estimated that by 2050 ca 21 million hip fractures would occur globally which will be leading cause of premature death and disability. Despite the need for improvement in the treatment for fracture repair, methods for treating fractures have changed little in recent decades. Systemic delivery of fracture-homing bone anabolics holds great promise as a therapeutic strategy in this regard. Here we report the design of a fracture-targeted peptide comprised of a payload that binds and activates the parathyroid hormone receptor (PTHR1) and is linked to a targeting ligand comprised of 20 D-glutamic acids (D-Glu20) that directs accumulation of the payload specifically at fracture sites. This targeted delivery results in reduction of fracture healing times to <1/2 while creating repaired bones that are >2-fold stronger than saline-treated controls in mice. Moreover, this hydroxyapatite-targeted peptide can be administered without detectable toxicity to healthy tissues or modification of healthy bones in dogs. Additionally, since similar results are obtained upon treatment of osteoporotic and diabetic fractures in mice, and pain resolution is simultaneously accelerated by this approach, we conclude that this fracture-targeted anabolic peptide displays significant potential to revolutionize the treatment of bone fractures.

1473-P: Combination Therapy with Dual-Function Peptide S1K and Insulin Reduces Blood Glucose Levels in Mouse Models of Type 1 Diabetes through Beta-Cell Regeneration and Immunosuppression

β cell regeneration is necessary to cure patients with type 1 diabetes (T1D) whose β cells are destroyed by immune cells. Although insulin therapy can control blood glucose levels, it must be administered throughout life and occurs several side effects such as hypoglycemia. New β cell can be generated through neogenesis, β cell replication or transdifferentiation from other cells in diabetic animals. In addition to β cell regeneration, immunosuppression is another important mechanism to prevent β cell destruction, but currently any drugs haven’t been approved as immunosuppressants for T1D treatments yet. A novel 9-mer peptide, Shiloah 1000 (S1K) , has dual functions of β cell regeneration and immunosuppression. In streptozotocin-induced hyperglycemic mouse model, the number of mice recovered to normoglycemia was increased by the combined treatment of S1K and insulin compared to S1K or insulin alone. In addition, the combined treatment more increased the number of β cells than S1K or insulin alone. Interestingly, insulin/glucagon double-positive cells were increased by both combined and S1K alone treatments. The increase in the number of mice recovered to normoglycemia is considered to be due to β cell regeneration through α- to β-cell transdifferentiation by S1K. In the NOD mouse model, the combined treatment of S1K and insulin suppressed hyperglycemia more than insulin alone. In addition, the serum C-peptide levels were increased by both combined and S1K treatments, but not by insulin treatment. The increase in the serum C-peptide level suggest that β cells remain undestroyed by immunosuppression. Taken together, our data show that S1K, as a drug candidate for T1D treatment in combination with insulin reduces blood glucose levels in the T1D mouse models through β cell regeneration and immunosuppression, suggesting a potential role in preventing and curing T1D. Disclosure Y. Lee: Employee; Ensolbiosciences. H. Kim: Employee; Ensolbiosciences. J. Kim: Employee; Ensol Bioscience. Y. Jang: Employee; Ensolbiosciences.

Novel active stealth micelles based on β2M achieved effective antitumor therapy

Micelles have been extensively investigated as drug delivery systems for loading of antitumor drugs with the advantages of good dispersibility, controllable size distribution, and high loading capacity. However, phagocytic clearance by the mononuclear phagocyte system remains a major impediment that inhibits blood circulation and thus tumor accumulation of micelles. Inspired by the antiphagocytic properties of β2-microglobulin (β2M), here we developed a β2M-derived peptide for the surface functionalization of micelles. A β2M-derived sequence was integrated with a matrix metalloproteinase-2 (MMP-2) cleavable sequence and then modified on the surface of poly(ethylene glycol)-b-poly(caprolactone) (PEG-PCL) micelles, endowing the micelles with both antiphagocytic and MMP-2-responsive properties. Compared to pristine PEG-PCL micelles, micelles modified with the dual-functional peptide exhibited higher inhibition of phagocytosis by macrophages in the absence of MMP-2, and enhanced internalization by tumor-associated macrophages in the presence of MMP-2, leading to enhanced tumor accumulation of the loaded photosensitizer, thus enabling antitumor therapy. These results demonstrated that antiphagocytic peptides derived from endogenous proteins are promising for functionalization of micelles in smart drug delivery.

Bioinspired core-shell silica nanoparticles monitoring extra- and intra-cellular drug release

Förster resonance energy transfer (FRET) has been widely used for monitoring drug release from nanoparticles (NPs). To understand the drug release from bioinspired drug-core silica-shell NPs, we synthesised two types of NPs using the dual-functional peptide SurSi via biosilicification for the first time, i.e., silica NP conjugated with FRET (Cy3 and Cy5) molecules, and FRET-core (DiO and DiI) silica-shell NP with different shell thicknesses (18 and 41 nm). The release kinetics of these two types of NPs were investigated under different conditions, including fetal bovine serum (FBS) and in cells, to mimic the drug release during blood circulation and intracellularly. Two different drug release mechanisms were identified. Cargo diffusion dominated the release during circulation, while the degradation of silica shell played a key role in drug release intracellularly.

Peptide-based colorimetric and fluorescent dual-functional probe for sequential detection of copper(Ⅱ) and cyanide ions and its application in real water samples, test strips and living cells

A novel dual-functional peptide probe FLH based on fluorescent “on–off–on” strategy and colorimetric visualization method was designed and synthesized. This new probe exhibited highly selective and rapid detection of Cu2+ with significant fluorescent “turn-off” response, with a visible colorimetric change from yellow to orange. The combination ratio of FLH to Cu2+ (1:1) was determined using ESI-HRMS spectra and Job’s plot. The fluorescent emission showed a good linear response (R2 = 0.9986) with a low detection limit of 1.5 nM. In addition, the FLH-Cu2+ complex displayed colorimetric changes and a fluorescent “off–on” response toward CN− over a wide pH range from 7 to 12. This detection behavior was observed within 20 s, with a limit of detection (LOD) for CN− at 12.7 nM. Based on stability and accuracy, FLH was next developed as dual-functional test strips, and was also successfully applied to detect Cu2+ and CN− in two actual water samples. More importantly, the cytotoxicity studies indicated that FLH had good biocompatibility and low toxicity, and was successfully utilized for monitoring Cu2+ and CN− in living cells through fluorescence imaging.

Angiotensin II As A Natural Suppressor On Cardiac Natriuretic Peptide System: Molecular Insights And Novel Therapeutic Design

<h3>Introduction</h3> Therapeutic strategies harnessing both renin-angiotensin system (RAS) and natriuretic peptide system (NPS) mediated effects, such as Sacubitril/Valsartan, has been proven to be promising in treating human heart failure (HF). However, molecular understanding of the interaction between RAS and NPS remains to be fully elucidated. While angiotensin II (ANGII), the central RAS hormone, is thought to interact with NPS at multiple levels, whether and how it may influence the actions of particulate guanylyl cyclase A (pGC-A), the only functional receptor for both atrial and b-type natriuretic peptide (ANP and BNP), has not been investigated. <h3>Hypothesis</h3> We hypothesize that ANGII naturally suppresses pGC-A and the inhibition of this crosstalk would be a novel therapeutic strategy to combat cardiovascular disease (CVD) of which RAS hyperactivation is a hallmark feature. <h3>Methods</h3> Plasma ANP, BNP, cGMP, and ANGII were measured in 128 healthy humans to define the balance between ANGII and NPS. cGMP responses to acute infusion of ANP alone and in the presence of ANGII was characterized in normal rats. The molecular mechanisms including involvement of ANGII type I receptor (AT1R) and its downstream target protein kinase C (PKC) were investigated in HEK293 cell overexpressing pGC-A and together with AT1R. CRRL-58, an innovative dual-functional peptide was designed to activate pGC-A and inhibit PKC, and tested for cGMP generation in HEK293 cells. <h3>Results</h3> In healthy human, plasma cGMP was positively associated with ANP or BNP only in low (≤ 4.5 pg/mL) but not those with high (> 4.5 pg/mL) ANGII levels (see Figure). Infusion of ANP (300 pmol/kg/min) in normal rats increased plasma and urinary cGMP, however ANP mediated cGMP generation was reduced in the presence of ANGII (50 pmol/kg/min). Notably, the suppressive effect of ANGII on pGC-A was recapitulated in HEK293 cell overexpressing both pGC-A and AT1R, but not in HEK293 cell overexpressing pGC-A alone. Meanwhile, we found PKC, a well-established downstream target of AT1R, was a key mediator for this observed crosstalk, as the suppression effect of ANGII was largely ablated by Go6983, a pan PKC inhibitor. Notably, CRRL-58 had superior potency in cGMP generation compared to the currently best-in-class ANP-analog, MANP, in vitro. <h3>Conclusions</h3> Our human and experimental data demonstrate that ANGII is a natural suppressor of pGC-A activation via AT1R and PKC signaling, but not via pGC-A per se. Moreover, we report for the first time that CRRL-58, an innovative bioengineered peptide that activates pGC-A receptor while inhibiting PKC signaling, potently generates cGMP. Together, our study provides new insights into the interaction between RAS and NPS, and highlights a novel and mechanism-based therapeutic avenue for treating HF and other CVDs.

Dextran and peptide-based pH-sensitive hydrogel boosts healing process in multidrug-resistant bacteria-infected wounds

Traumatic multidrug-resistant (MDR) bacterial infections are deadly threat to the public. To combat MDR bacteria, we developed a dual functional pH-sensitive hydrogel based on peptide DP7 (VQWRIRVAVIRK) and oxidized dextran (DP7-ODEX hydrogel). As an antimicrobial peptide, DP7 can synergize with many antibiotics; thus, we loaded ceftazidime into DP7-ODEX hydrogel, which showed an obvious advantage in MDR P. aeruginosa inhibition. Additionally, due to the interaction between aldehyde groups in oxidized dextran and amine groups from wound tissue, the hydrogel could extend on the irregular surface of skin defects and promote epithelial cells adhesion. DP7 could also be used as a wound-healing peptide and accelerate the healing process. We confirmed that the DP7-ODEX hydrogel exerted formidable therapeutic effects in normal or diabetic wound infection model. According to histomorphology analysis we found that DP7 hydrogel also have a scarless wound healing ability. In summary, we developed a hydrogel fabricated by the dual functional peptide DP7 that can kill multidrug-resistant bacteria colonizing the wound bed and boost scarless wound healing.

Abstract 10303: CRRL290: A Novel and Mechanism-Based Therapeutic Peptide Targeting the Crosstalk Between Angiotensin II and the Natriuretic Peptide System for Cardiorenal Disease

Introduction: Therapeutic strategies dual-targeting renin-angiotensin system (RAS) and natriuretic peptide system (NPS), such as Sacubitril/Valsartan, have been proven promising in treating cardiorenal disease (CRD). However, mechanisms behind the crosstalk between RAS and NPS remain to be fully elucidated. While angiotensin II (ANGII), the central RAS hormone, is thought to interact with NPS at multiple levels, whether and how it may influence the actions of particulate guanylyl cyclase A (pGC-A), the receptor activated by ANP and BNP to increase its second messenger cGMP, has not been investigated. Hypothesis: We hypothesize ANGII naturally suppresses pGC-A activity and inhibition of this ANGII mediated action can be a new strategy to combat CRD. Methods: Studies consist of biomarker analyses in plasma from healthy humans, drug infusions in normal rats, molecular characterization in human HEK293 cells, and in vitro assessment of CRRL290, an innovative dual-functional peptide designed to activate pGC-A and inhibit protein kinase C (PKC). Results: In 128 healthy humans, plasma cGMP was positively associated with ANP or BNP in low (≤ 4.5 pg/mL) but not those with high (&gt; 4.5 pg/mL) ANGII levels. Infusion of ANP (300 pmol/kg/min) in normal rats increased plasma and urinary cGMP, however ANP mediated cGMP generation was reduced in the presence of ANGII. Interestingly, suppression effect of ANGII on pGC-A was only recapitulated in HEK293 cell overexpressing both pGC-A and AT1R, but not in HEK293 cell overexpressing pGC-A alone. We also found PKC, a known downstream target of AT1R, was a key mediator as the suppression effect of ANGII was largely ablated by Go6983, a pan PKC inhibitor. Accordingly, we designed and synthesized CRRL290. Notably, CRRL290 had superior potency in cGMP generation compared to the currently best-in-class ANP-analog, MANP, in HEK293 cells. Conclusions: We demonstrate that ANGII is a natural suppressor of pGC-A via AT1R and PKC signaling. We also report for the first time that CRRL290, a novel bioengineered peptide that activates pGC-A receptor while inhibiting PKC signaling, potently generates cGMP. Our study provides new insights into the RAS-NPS interaction, and highlights a novel and mechanism-based therapeutic avenue for treating CRD.

In Silico Screening of a Bile Acid Micelle Disruption Peptide for Oral Consumptions from Edible Peptide Database.

Recently, many bioactive peptides have been identified using bioinformatics tools. Previously, our group developed a method to screen dual-functional peptides that have direct intestinal delivery with porous silica gel and bile acid micelle disruption. However, newly designed peptides were not found in any storage protein. Therefore, in this study, in silico screening was performed using a 350,000 edible peptide library consisting of 4- to 7-mer independent peptides. As an initial screening, all edible peptides were applied to the random forest model to select predicted positive peptides. For a second screening, the peptides were assessed for the possibility of intestinal delivery using a 3D color map. From this approach, three novel dual-functional peptides, VYVFDE, WEFIDF, and VEEFYC were identified, and all of them were derived from storage proteins (legumin, myosin, and 11S globulin). In particular, VEEFYCS, in which a serine residue (S) is added to VEEFYC, was assumed to be released by thermolysin from the 11S-globulin derived from Ginkgo biloba by LC-MS/MS analysis. VEEFYCS was found to have suitable direct intestinal delivery and bile acid micelle disruption activity.

Continuous stimulation of dual-function peptide PGLP-1-VP inhibits the morbidity and mortality of NOD mice through anti-inflammation and immunoregulation

Multiple animal and human studies have shown that administration of GLP-1RA can enhance β-cell recovery, reduce insulin dosage, reduce HbA1c content in the blood, reduce the risk of hypoglycemia and reduce inflammation. In the NOD mouse model, peptide VP treatment can prevent and treat type 1 diabetes through immunomodulation. Therefore, we designed a new dual-functional PGLP-1-VP, which is expected to combine the anti-inflammatory effect of PGLP-1 and the immunomodulatory effect of VP peptide. In streptozotocin-induced hyperglycemic mice model, we demonstrated that PGLP-1-VP can act as a GLP-1R agonist to improve hyperglycemia and increase insulin sensitivity. In the NOD mouse model, PGLP-1-VP treatment reduced morbidity, mortality, and pancreatic inflammation, and showed superior effect to PGLP-1 or VP treatment alone, confirming that PGLP-1-VP may act as a dual-function peptide. PGLP-1-VP provided immunomodulatory effect through increasing Th2 cell percentage and balancing the ratio of Th2/Th1 in spleen and PLN, similar to P277 and VP. Additionally, PGLP-1-VP and PGLP-1 act the anti-inflammation by increasing Treg cells and TGF-β1 content like DPP-IV inhibitor. Taken together, our data shows that the dual-functional PGLP-1-VP reduces morbidity and mortality in the NOD model, suggesting a potential role in preventing and treating type 1 diabetes.

Design and screening of a novel neuropilin-1 targeted penetrating peptide for anti-angiogenic therapy in glioma

AimsThis study aimed to design and screen a dual functional fusion peptide that could penetrate the blood-brain barrier and target neuropilin 1 (NRP1) overexpressed in vascular endothelial cells for the anti-angiogenesis of glioma treatment. Main methodsAt the cellular level, the in vitro anti-angiogenic activity of six NRP1 targeting peptides was screened by testing the ability to inhibit the proliferation and tube formation of HUVECs. Then, the in vitro anti-angiogenic activity of two fusion peptides containing different linkers was screened by testing the ability to inhibit HUVECs proliferation, tube formation and migration. The effect of fusion peptide on VEGFR2 related signal pathway was confirmed by Western-blotting. Surface plasmon resonance technology was used to detect the affinity of the fusion peptide to NRP1. The ability of FITC-labeled peptides to penetrate cells was confirmed by cell uptake assay. By establishing an orthotopic glioma model, we evaluated the ability of FITC-labeled peptides to penetrate the blood-brain barrier and their anti-glioma growth activity in vivo. Key findingsWe found that NRP1 targeting peptide RP7 and linker cysteine were the most suitable key components in the fusion peptide. We also found that the fusion peptide Tat-C-RP7 we constructed had the strongest ability to penetrate the blood-brain barrier and anti-angiogenic activity in vitro and in vivo. SignificanceAt present, NRP1 targeting peptide as a drug delivery tool and molecular probe seems to have received more attention. We constructed a fusion peptide Tat-C-RP7 with strong anti-angiogenic activity for the treatment of glioma.