Bifunctional Peptide Research Articles

Abiotic Acyl Transfer Cascades Driven by Aminoacyl Phosphate Esters and Self-Assembly.

Biochemical acyl transfer cascades, such as those initiated by the adenylation of carboxylic acids, are central to various biological processes, including protein synthesis and fatty acid metabolism. Designing cascade reactions in aqueous media remains challenging due to the need to control multiple, sequential reactions in a single pot and manage the stability of reactive intermediates. Herein, we developed abiotic cascades using aminoacyl phosphate esters, the synthetic counterparts of biological aminoacyl adenylates, to drive sequential chemical reactions and self-assembly in a single pot. We demonstrated that the structural elements of amino acid side chains (aromatic versus aliphatic) significantly influence the reactivity and half-lives of aminoacyl phosphate esters, ranging from hours to days. This behavior, in turn, affects the number of couplings we can achieve in the network and the self-assembly propensity of activated intermediate structures. The cascades are constructed using bifunctional peptide substrates featuring side chain nucleophiles. Specifically, aromatic amino acids facilitate the formation of transient thioesters, which preorganized into spherical aggregates and further couple into chimeric assemblies composed of esters and thioesters. In contrast, aliphatic amino acids, which lack the ability to form such structures, predominantly undergo hydrolysis, bypassing further transformations after thioester formation. Additionally, in mixtures containing multiple aminoacyl phosphate esters and peptide substrates, we achieved selective product formation by following a distinct pathway that favors subsequent reactions through reactivity changes and self-assembly. By coupling chemical reactions with molecules of varying reactivity time scales, we can drive multiple reaction clocks with distinct lifetimes and self-assembly dynamics, facilitating precise temporal and structural regulation.

Fully synthetic, tunable poly(α-amino acids) as the base of bioinks curable by visible light

Bioinks play a crucial role in tissue engineering, influencing mechanical and chemical properties of the printed scaffold as well as the behavior of encapsulated cells. Recently, there has been a shift from animal origin materials to their synthetic alternatives. In this context, we present here bioinks based on fully synthetic and biodegradable poly(α,L-amino acids) (PolyAA) as an alternative to animal-based gelatin methacrylate (Gel-Ma) bioinks. Additionally, we first reported the possibility of the visible light photoinitiated incorporation of the bifunctional cell adhesive RGD peptide into the PolyAA hydrogel matrix. The obtained hydrogels are shown to be cytocompatible, and their mechanical properties closely resemble those of gelatin methacrylate-based scaffolds. Moreover, combining the unique properties of PolyAA-based bioinks, the photocrosslinking strategy, and the use of droplet-based printing allows the printing of constructs with high shape fidelity and structural integrity from low-viscosity bioinks without using any sacrificial components. Overall, presented PolyAA-based materials are a promising and versatile toolbox that extends the range of bioinks for droplet bioprinting.

Crystal structure of Trichinella spiralis calreticulin and the structural basis of its complement evasion mechanism involving C1q.

Helminths produce calreticulin (CRT) to immunomodulate the host immune system as a survival strategy. However, the structure of helminth-derived CRT and the structural basis of the immune evasion process remains unclarified. Previous study found that the tissue-dwelling helminth Trichinella spiralis produces calreticulin (TsCRT), which binds C1q to inhibit activation of the complement classical pathway. Here, we used x-ray crystallography to resolve the structure of truncated TsCRT (TsCRTΔ), the first structure of helminth-derived CRT. TsCRTΔ was observed to share the same binding region on C1q with IgG based on the structure and molecular docking, which explains the inhibitory effect of TsCRT on C1q-IgG-initiated classical complement activation. Based on the key residues in TsCRTΔ involved in the binding activity to C1q, a 24 amino acid peptide called PTsCRT was constructed that displayed strong C1q-binding activity and inhibited C1q-IgG-initiated classical complement activation. This study is the first to elucidate the structural basis of the role of TsCRT in immune evasion, providing an approach to develop helminth-derived bifunctional peptides as vaccine target to prevent parasite infections or as a therapeutic agent to treat complement-related autoimmune diseases.

Abstract 7255: Peptide-based siRNA delivery for tumor-specific targeting of lactate dehydrogenase C

Abstract Background: Breast cancer is the most common cancer worldwide and the leading cause of female cancer-related deaths, highlighting the pressing need for novel therapeutic interventions. Treatment of triple negative breast cancer in particular remains a challenge due to disease aggressiveness and the lack of durable treatment options. We speculate that targeting of Lactate Dehydrogenase C (LDHC) could be a novel approach given its highly tumor specific expression and immunogenic properties. We recently demonstrated that LDHC acts as a key regulator of the DNA damage response and tumor mitotic fidelity. Moreover, silencing of LDHC significantly improved treatment response to DNA damage response related anti-cancer drugs, highlighting its therapeutic potential for cancer precision medicine. Methods: We characterized and evaluated several cell penetrating peptide-siRNA complexes for efficient silencing of LDHC in tumor cells using a 10R polyarginine peptide or bifunctional 10R-RGD, cRGD-10R or iRGD-10R peptides with tumor homing and penetrating abilities. Results: Peptide-siRNA complex formation and tumor cellular uptake was confirmed using gel retardation assays and fluorescent confocal imaging. Biophysiochemical characterization and transmission electron microscopy revealed the complexes to be uniform in size with a low polydispersity index and positive zeta potentials. LDHC silencing in breast cancer cells using the peptide complexes demonstrated a 30-53% reduction in LDHC expression, with the 10R-siRNA and cRGD-10R-siRNA complexes showing the highest silencing efficiency. In 3D breast cancer spheroids, transfection with the 10R-siRNA complex resulted in a 20% downregulation of LDHC expression. Further analysis of the bifunctional-siRNA complexes in 3D cultures is ongoing as well as cytotoxicity analyses on non-cancerous cell lines. Conclusions: Our findings demonstrate that peptide-mediated delivery of LDHC siRNA can reduce LDHC expression in 2D and 3D breast cancer cultures, suggesting that they could be used as a valuable approach for therapeutic intervention. Citation Format: Adviti Naik, Hanan Qasem, Julie Decock. Peptide-based siRNA delivery for tumor-specific targeting of lactate dehydrogenase C [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7255.

Bifunctional peptides as alternatives to copper-based formulations to control citrus canker

Citrus canker is an infectious bacterial disease and one of the major threats to the orange juice industry, a multibillion-dollar market that generates hundreds of thousands of jobs worldwide. This disease is caused by the Gram-negative bacterium Xanthomonas citri subsp. citri. In Brazil, the largest producer and exporter of concentrate orange juice, the control of citrus canker is exerted by integrated management practices, in which cupric solutions are intensively used in the orchards to refrain bacterial spreading. Copper ions accumulate and are as heavy metals toxic to the environment. Therefore, the aim of the present work was to evaluate bifunctional fusion proteins (BiFuProts) as novel and bio-/peptide-based alternatives to copper formulations to control citrus canker. BiFuProts are composed of an anchor peptide able to bind to citrus leaves, and an antimicrobial “killer” peptide to protect against bacterial infections of plants. The selected BiFuProt (Mel-CgDEF) was bactericidal against X. citri at 125 μg mL−1, targeting the bacterial cytoplasmic membrane within the first minutes of contact. The results in the greenhouse assays proved that Mel-CgDEF at 250 μg mL−1 provided protection against X. citri infection on the leaves, significantly reducing the number of lesions by area when compared with the controls. Overall, the present work showed that the BiFuProt Mel-CgDEF is a biobased and biodegradable possible alternative for substitute cupric formulations.Key points• The bifunctional fusion protein Mel-CgDEF was effective against Xanthomonas citri.• Mel-CgDEF action mechanism was the disruption of the cytoplasmic membrane.• Mel-CgDEF protected citrus leaves against citrus canker disease.

Functionalization of Bacterial Cellulose with the Antimicrobial Peptide KR-12 via Chimerical Cellulose-Binding Peptides.

Bacterial-derived cellulose (BC) has been studied as a promising material for biomedical applications, including wound care, due to its biocompatibility, water-holding capacity, liquid/gas permeability, and handleability properties. Although BC has been studied as a dressing material for cutaneous wounds, to date, BC inherently lacks antibacterial properties. The current research utilizes bifunctional chimeric peptides containing carbohydrate binding peptides (CBP; either a short version or a long version) and an antimicrobial peptide (AMP), KR-12. The secondary structure of the chimeric peptides was evaluated and confirmed that the α-helix structure of KR-12 was retained for both chimeric peptides evaluated (Long-CBP-KR12 and Short-CBP-KR12). Chimeric peptides and their individual components were assessed for cytotoxicity, where only higher concentrations of Short-CBP and longer timepoints of Short-CBP-KR12 exposure exhibited negative effects on metabolic activity, which was attributed to solubility issues. All KR-12-containing peptides exhibited antibacterial activity in solution against Escherichia coli (E. coli) and Pseudomonas aeruginosa (P. aeruginosa). The lipopolysaccharide (LPS) binding capability of the peptides was evaluated and the Short-CBP-KR12 peptide exhibited enhanced LPS-binding capabilities compared to KR-12 alone. Both chimeric peptides were able to bind to BC and were observed to be retained on the surface over a 7-day period. All functionalized materials exhibited no adverse effects on the metabolic activity of both normal human dermal fibroblasts (NHDFs) and human epidermal keratinocyte (HaCaT) epithelial cells. Additionally, the BC tethered chimeric peptides exhibited antibacterial activity against E. coli. Overall, this research outlines the design and evaluation of chimeric CBP-KR12 peptides for developing antimicrobial BC membranes with potential applications in wound care.

Bi-functional KIT-PR1P peptides combine with VEGF to protect ischemic kidney in rats by targeting to Kim-1

IntroductionAcute kidney injury (AKI) was a disease with a high mortality mainly caused by renal ischemia/reperfusion injury (I/R). Although the current non-targeted administration of vascular endothelial growth factor (VEGF) for AKI had been revealed to facilitate the recovery of renal I/R, how to targeted deliver VEGF and to retain it efficiently in the ischemic kidney was critical for its clinical application. MethodsIn present study, bi-functional KIT-PR1P peptides were constructed which bond VEGF through PR1P domain, and targeted ischemic kidney through KIT domain to interact with biomarker of AKI-kidney injury molecule-1 (Kim-1). Then the targeted and therapeutic effects of KIT-PR1P/VEGF in AKI was explored in vitro and in vivo. ResultsThe results showed KIT-PR1P exhibited better angiogenic capacity and targeting ability to hypoxia HK-2 cells with up-regulated Kim-1 in vitro. When KIT-PR1P/VEGF was used for the treatment of renal I/R through intravenous administration in vivo, KIT-PR1P could guide VEGF and retain its effective concentration in ischemic kidney. In addition, KIT-PR1P/VEGF promoted angiogenesis, alleviated renal tubular injury and fibrosis, and finally promoted functional recovery of renal I/R. ConclusionThese results indicated that the bi-functional KIT-PR1P peptides combined with VEGF would be a promising strategy for the treatment of AKI by targeting to Kim-1.

Pre-clinical evaluation of biomarkers for the early detection of nephrotoxicity following alpha-particle radioligand therapy.

Cancer treatment with alpha-emitter-based radioligand therapies (α-RLTs) demonstrates promising tumor responses. Radiolabeled peptides are filtered through glomeruli, followed by potential reabsorption of a fraction by proximal tubules, which may cause acute kidney injury (AKI) and chronic kidney disease (CKD). Because tubular cells are considered the primary site of radiopeptides' renal reabsorption and potential injury, the current use of kidney biomarkers of glomerular functional loss limits the evaluation of possible nephrotoxicity and its early detection. This study aimed to investigate whether urinary secretion of tubular injury biomarkers could be used as an additional non-invasive sensitive diagnostic tool to identify unrecognizable tubular damage and risk of long-term α-RLT nephrotoxicity. A bifunctional cyclic peptide, melanocortin 1 ligand (MC1L), labeled with [203Pb]Pb-MC1L, was used for [212Pb]Pb-MC1L biodistribution and absorbed dose measurements in CD-1 Elite mice. Mice were treated with [212Pb]Pb-MC1L in a dose-escalation study up to levels of radioactivity intended to induce kidney injury. The approach enabled prospective kidney functional and injury biomarker evaluation and late kidney histological analysis to validate these biomarkers. Biodistribution analysis identified [212Pb]Pb-MC1L reabsorption in kidneys with a dose deposition of 2.8, 8.9, and 20 Gy for 0.9, 3.0, and 6.7 MBq injected [212Pb]Pb-MC1L doses, respectively. As expected, mice receiving 6.7 MBq had significant weight loss and CKD evidence based on serum creatinine, cystatin C, and kidney histological alterations 28 weeks after treatment. A dose-dependent urinary neutrophil gelatinase-associated lipocalin (NGAL, tubular injury biomarker) urinary excretion the day after [212Pb]Pb-MC1L treatment highly correlated with the severity of late tubulointerstitial injury and histological findings. Urine NGAL secretion could be a potential early diagnostic tool to identify unrecognized tubular damage and predict long-term α-RLT-related nephrotoxicity.

Bifunctional Peptide Nanofibrils for Targeted Protein Degradation.

Proteolysis targeting chimera (PROTAC) is a state-of-the-art technology for ablating undruggable targets. A PROTAC degrader achieves targeted protein degradation (TPD) through the simultaneous binding of a protein of interest (POI) and an E3 ligase to form a ternary complex. A nanofibril-based PROTAC strategy to form a polynary (E3)m : PROTAC : (POI)n complex has not been reported in the TPD field up to this point. A recent innovation shows that a POI ligand and E3 ligase ligand don't have to be within a fused degrader molecule. Instead, they can be recruited to cellular proximity by a self-assembly-driving peptide and click chemistry. The resulting nanofibrils can recruit multiple POI and E3 ligase molecules to form a polynary complex as a degradation center. The so-called Nano-PROTAC provides a novel approach for TPD in cancer therapy.

Bifunctional Peptide Nanofibrils for Targeted Protein Degradation

AbstractProteolysis targeting chimera (PROTAC) is a state‐of‐the‐art technology for ablating undruggable targets. A PROTAC degrader achieves targeted protein degradation (TPD) through the simultaneous binding of a protein of interest (POI) and an E3 ligase to form a ternary complex. A nanofibril‐based PROTAC strategy to form a polynary (E3)m : PROTAC : (POI)n complex has not been reported in the TPD field up to this point. A recent innovation shows that a POI ligand and E3 ligase ligand don't have to be within a fused degrader molecule. Instead, they can be recruited to cellular proximity by a self‐assembly‐driving peptide and click chemistry. The resulting nanofibrils can recruit multiple POI and E3 ligase molecules to form a polynary complex as a degradation center. The so‐called Nano‐PROTAC provides a novel approach for TPD in cancer therapy.

Abstract B046: A first-in-human Phase 1a/b of PEP-010, a proapoptotic bifunctional peptide, administered as single agent and in combination with paclitaxel in patients with recurrent and/or metastatic solid cancer: results from the dose escalation study

Abstract Background: PEP-010 is a first-in-class bifunctional peptide that penetrates cells and specifically disrupts the interaction between Caspase9 and PP2A, two proteins involved in the apoptotic cascade, leading to Caspase9-dependent apoptosis in cancer cells. Safety, PK, and preliminary anti-tumor activity from the dose escalation part of a Phase 1a/b study of PEP-010 as a monotherapy and in combination with paclitaxel (P) in recurrent and/or metastatic solid cancer are presented. Methods: PEP-010 was administered as a 3-hour intravenous weekly infusion on 3 consecutive days in patients (pts) with advanced cancer ineligible for standard therapy, in monotherapy (Arm A) or in combination with weekly P (Arm B) for pts eligible for P. In Arm A, pts were enrolled into 8 dose cohorts starting at 0.15 mg/kg up to 15 mg/kg, using an accelerated titration design (first 4 dose levels (DLs)), followed by a 3+3 design. In Arm B, 5 DLs of PEP-010 combined with weekly P (80mg/m²) were explored. The primary objective was the determination of the maximum tolerated dose (MTD) and the recommended phase 2 dose (RP2D) of PEP-010 in both arms, based on occurrence of dose-limiting toxicities (DLTs). Secondary objectives were the safety and tolerability profile, PK and preliminary antitumor activity assessed by RECIST1.1. Results: At data cutoff (March 7, 2023), 19 (Arm A) and 15 (Arm B) pts received PEP-010 across 8 and 3 DLs, respectively. Median age was 57 [32-78] years and 56% were female. Median number of prior therapies was 6 [2-13]. A DLT (Grade 2 infusion-related reaction (IRR)) was observed in 1 pt in Arm A at DL 8 (15 mg/kg). In Arm B, 3 pts experienced a DLT at DL 5 mg/kg: 1 Grade 4 neutropenia and 2 IRR (inacceptable Grade 2 and 4). All DLTs led to permanent treatment discontinuation. MTD was defined as 15 mg/kg in Arm A and 2.5 mg/kg in Arm B. Most common adverse events (AE) related to PEP-010 included asthenia (26.5%) and IRR (14.7%) whereas most common AEs related to P included anemia (66.7%), peripheral neuropathy (33.3%), neutropenia (20%), onychopathy (20%), and diarrhea (20%). Median time on treatment was 5.1 weeks [2.1-25.3] in Arm A and 5.3 [3-18] in Arm B. In Arm A, for 7 pts (36.8%) a stable disease (SD) was observed for a median of 2.8 months [0.6-4.5]. In Arm B, 4 (26.7%) confirmed partial responses were observed in pts with ovarian, pancreatic and thyroid adenocarcinomas, and squamous cell lung carcinoma, and 2 pts (13.3%) had SD for a median of 4 months [2.7-9.6]. PK data indicated a proportionally dose-related increase of systemic exposure to PEP-010. No significant accumulation of PEP-010 over time or impact of P combination was observed. Conclusions: Data from the Escalation Part of the Phase 1a/b clinical trial showed an acceptable safety profile of PEP-010 in monotherapy. In combination, the dose of PEP-010 could not be escalated to the same level because of IRR. Preliminary antitumoral activity was observed with PEP-010 in combination with P including in pts who had previously received taxanes. Further clinical development is warranted. Citation Format: Pauline Du Rusquec, Elodie Coquan, Frederique Berger, Keyvan Rezai, Pierre-Emmanuel Brachet, Sophie Postel-Vinay, Zahra Castel-Ajgal, Gregoire Marret, Marie-Paule Sablin, Segolene Hescot, Raphael Sanchez, Anne-Claire Coyne, Claire Bertrand, Clementine Bouchez, Elsa Lafitte, Lucas Frezouls, Carole Mardesson, Abdelkrim Taamma, Chantal Krezel, Laura Dadon, Christophe Le Tourneau. A first-in-human Phase 1a/b of PEP-010, a proapoptotic bifunctional peptide, administered as single agent and in combination with paclitaxel in patients with recurrent and/or metastatic solid cancer: results from the dose escalation study [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B046.

Construction of multifunctional coating with cationic amino acid-coupled peptides for osseointegration of implants

Osseointegration is an important indicator of implant success. This process can be improved by coating modified bioactive molecules with multiple functions on the surface of implants. Herein, a simple multifunctional coating that could effectively improve osseointegration was prepared through layer-by-layer self-assembly of cationic amino acids and tannic acid (TA), a negatively charged molecule. Osteogenic growth peptide (OGP) and the arginine-glycine-aspartic acid (RGD) functional polypeptides were coupled with Lys6 (K6), the two polypeptides then self-assembled with TA layer by layer to form a composite film, (TA-OGP@RGD)n. The surface morphology and biomechanical properties of the coating were analyzed in gas and liquid phases, and the deposition process and kinetics of the two peptides onto TA were monitored using a quartz crystal microbalance. In addition, the feeding consistency and adsorption ratios of the two peptides were explored by using fluorescence visualization and quantification. The (TA-OGP@RGD)n composite membrane mediated the early migration and adhesion of cells and significantly promoted osteogenic differentiation and mineralization of the extracellular matrix in vitro. Additionally, the bifunctional peptide exhibited excellent osteogenesis and osseointegration owing to the synergistic effect of the OGP and RGD peptides in vivo. Simultaneously, the (TA-OGP@RGD)n membrane regulated the balance of reactive oxygen species in the cell growth environment, thereby influencing the complex biological process of osseointegration. Thus, the results of this study provide a novel perspective for constructing multifunctional coatings for implants and has considerable application potential in orthopedics and dentistry.

Efficient titanium surface modified using bifunctional chimeric peptides to prevent biofilm formation by multiple microorganisms.

It is still a challenge to prevent the formation of bacterial biofilms on the surfaces of oral implants. A chemical peptide with binding and antibacterial properties may be a promising agent if used to modify titanium (Ti) surfaces to inhibit biofilm formation. In this study, peptides were designed by linking the antimicrobial sequence derived from human β-defensin-3 (hBD-3) to the Ti-binding peptide-1 (TBP-1) sequence by using a triple glycine (G) linker. The antimicrobial activity and biocompatibility characteristics of the chemical-peptide-modified Ti surface were then evaluated and the potential antibacterial mechanism was investigated. This study demonstrated that the chemical-peptide-modified surface exhibited satisfactory bactericidal activities against Streptococcus gordonii, Fusobacterium nucleatum, and Porphyromonas gingivalis. In addition to its potent bacteria-killing efficacy, the surface-immobilised chemical peptide also demonstrated excellent biocompatibility to L929 cells. Moreover, the disruption of the integrity of the bacterial membrane partially revealed the antibacterial mechanism of the peptide. This study demonstrated the potential of chemical-peptide-modified Ti surfaces for preventing the occurrence of peri-implant diseases, thereby providing a promising approach to improving the survival rate of oral implants.

High pressure-assisted enzymatic hydrolysis promotes the release of a bi-functional peptide from cowhide gelatin with dipeptidyl peptidase IV (DPP-IV) inhibitory and antioxidant activities

The process of generating functional peptides from cowhide gelatin is challenged by inefficient enzymatic hydrolysis. In this study, the researchers attempted to enhance the hydrolysis and potential functional properties of the peptides by subjecting the cowhide gelatin to high-pressure treatment (200, 300, and 400 MPa) for 20 min, followed by enzymatic hydrolysis. The highest 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity, 2,2′ azinobis(3 ethylbenzothiazoline 6 sulfonic acid) (ABTS) radical scavenging activity, and DPP-IV inhibitory activity of the hydrolysate were obtained at 200 MPa, accompanied with an increase in the content of hydrophobic, acidic, and basic amino acids (P < 0.05). Correspondingly, the high-pressure pretreatment (200 MPa) reduced the thermal stability, particle size, and morphological integrity of cowhide gelatin, with a corresponding increase in the exposure of hydrophobic regions. Altogether, these results indicated that appropriate high-pressure-assisted enzymatic hydrolysis reinforced the release of bi-functional peptides by modifying the structure of cowhide gelatin.

Targeted Degradation of Alpha-Synuclein by Autophagosome-Anchoring Chimera Peptides.

Targeted protein degradation (TPD) confers knockdown of "undruggable" targets such as alpha-synuclein (αSyn), a pathogenic protein in multiple neurodegenerative diseases. Though many of these proteins were mainly degraded through the autophagy-lysosome pathway (ALP), few TPD tools harnessing the ALP were reported. Herein, we developed a strategy termed autophagosome-anchoring chimera (ATACC), in which the protein of interest (POI) can be anchored to microtubule-associated protein-1 light chain-3B (LC3B) on the autophagosome with the assistance of an LC3-interacting region (LIR)-containing bifunctional peptide, and the selective autophagy of the POI is thus facilitated. A series of αSyn-targeting ATACC peptides were designed and synthesized. Biological evaluations demonstrated that these compounds could degrade αSyn specifically and effectively through a "chemical-induced cargo recognition-ALP degradation" mechanism. The neuroprotective effects of ATACC peptide P1 were further validated in vitro and in vivo. Collectively, our results provided a new TPD tool and revealed a potential therapeutic strategy against synucleinopathies.

Transepithelial Transport of the Bifunctional Peptide IPYWTY Indirectly Induced Insulin Release Mediated by Active GLP-1.

There is currently no appropriate cell model suitable for evaluating the insulinotropic effects of DPP-4 inhibitory peptides (DPP-4IPs) mediated by active glucagon-like peptide-17-36 (active GLP-1). The study aims to evaluate the transepithelial transport of IPYWTY on its in situ insulinotropic effects by using a 2D and dual-layered coculture cell model that consists of Caco-2 and NCI-H716 cells on the apical (AP) side and β-TC-6 cells on the basolateral (BL) side. During transportation, IPYWTY was absorbed in its intact form through PepT1 and paracellular transport. Meanwhile, it was degraded to several peptide fragments, including PYWTY, YWTY, WTY, and IPY, which decreased its in situ DPP-4 inhibitory activity. IPYWTY does not directly stimulate insulin release in β-TC-6 cells, while it increased the active GLP-1 level from 76.57 ± 15.16 to 95.63 ± 1.99 pM (1.25 times) in NCI-H716 cells. Interestingly, IPYWTY indirectly increased insulin levels from 426.91 ± 6.07 to 573.94 ± 2.97 μIU/mL (1.34 times) in the 2D and dual-layered coculture cell model for its dual function of stimulating active GLP-1 secretion and DPP-4 inhibition. These results suggested that the 2D and dual-layered coculture cell model is an alternative strategy for effectively evaluating the insulinotropic effects of DPP-4IPs mediated by active GLP-1.

A chemoimmunotherapy-based strategy to enhance tumor therapy by cross-linking a novel size-variable nanocluster via a bifunctional peptide

The combination of chemotherapy and immunotherapy plays a synergistic role in improving the effectiveness of cancer treatment. However, it is a great challenge to deliver drugs accurately and efficiently to the target and exert synergistic effects due to multiple biological obstacles in vivo. Here, we constructed a novel size-variable three-dimensional mesh nanocluster (HA/P-A cluster) cross-linked by bifunctional peptide R14 for tumor targeted co-delivery of doxorubicin (DOX) and pPD-L1 trap. The ERP effect and the targeting effect of the outer layer of HA synergistically mediated the accumulation of nanoclusters in tumor tissues. Subsequently, R14 was broken by matrix metalloproteinase-2 (MMP-2) and the nanoclusters (180 nm) were cleaved into ultra-small size nanoparticles (∼10 nm), achieving deep penetration while targeting tumor cells through RGD at both ends of R14. Based on this strategy, DOX acts as an initiator to induce immunogenic cell death (ICD) while immunodrugs block the PD-L1 pathway. Both in vitro and in vivo studies have shown that HA/P-A cluster effectively retarded tumor growth. Moreover, HA/P-A cluster treatment resulted in a significant increased CD8+ T cell infiltration in tumor tissue. In conclusion, this strategy provides a new direction for targeted co-delivery of nanoplatforms to improve chemoimmunotherapy in solid tumors.

Structural Analysis of Non-native Peptide-Based Catalysts Using 2D NMR-Guided MD Simulations.

Proteins and enzymes generally achieve their functions by creating well-defined 3D architectures that pre-organize reactive functionalities. Mimicking this approach to supramolecular pre-organization is leading to the development of highly versatile artificial chemical environments, including new biomaterials, medicines, artificial enzymes, and enzyme-like catalysts. The use of β-turn and α-helical motifs is one approach that enables the precise placement of reactive functional groups to enable selective substrate activation and reactivity/selectivity that approaches natural enzymes. Our recent work has demonstrated that helical peptides can serve as scaffolds for pre-organizing two reactive groups to achieve enzyme-like catalysis. In this study, we used CYANA and AmberTools to develop a computational approach for determining how the structure of our peptide catalysts can lead to enhancements in reactivity. These results support our hypothesis that the bifunctional nature of the peptide enables catalysis by pre-organizing the two catalysts in reactive conformations that accelerate catalysis by proximity. We also present evidence that the low reactivity of monofunctional peptides can be attributed to interactions between the peptide-bound catalyst and the helical backbone, which are not observed in the bifunctional peptide.

Osteoclast-targeted delivery of anti-miRNA oligonucleotides by red blood cell extracellular vesicles.

Osteoporosis (OP) affects millions worldwide but currently cannot be cured. Suppressing the level of miR-214 in osteoclasts by the anti-miRNA oligonucleotide (AMO) anti-miR-214 reverses bone absorption and provides a potential treatment. Here we report a peptide-guided delivery strategy using red blood cell extracellular vesicles (RBCEVs) as the vehicle to realize osteoclast-targeted delivery of anti-miR-214. A bi-functional peptide, TBP-CP05, which binds to both the CD63 on RBCEVs and receptors on osteoclasts, acts as the guide. TBP-CP05 binds with RBCEVs through CP05, displays the TRAP-binding peptide (TBP) on the surface of EVs, and endows RBCEVs with osteoclast-targeting capability both in vitro and in vivo. Intravenous injection of the osteoclast-targeting RBCEVs (OT-RBCEVs) led to the enrichment of EVs in the bone skeleton, significant inhibition of the osteoclast activity, elevated osteoblast activity, and improved bone density in osteoporotic mice. Altogether, this work demonstrates efficient guidance of drug-loaded EVs to the targeted cells in vivo using bi-functional fusion peptides, and showcases that targeted delivery of anti-miR-214 by OT-RBCEVs may be a viable method for OP treatment. SIGNIFICANCE STATEMENT. Surface functionalization of EVs endows these nanovesicles cell-specific targeting property which guides the drug cargos to specific tissues and cells with higher accuracy, longer retention, and minimal off-target effects. Methods to functionalize EVs with minimal procedures are highly desired for clinical applications. Here we present a facile method using a bifunctional fusion peptide to guide RBCEVs to osteoclasts. A simple incubation of the bifunctional peptide and RBCEVs results in osteoclast-targeting RBCEVs (OT-RBCEVs) that effectively deliver anti-miR-214 to osteoclasts in vivo in a mouse model of osteoporosis, bringing a potential therapy to osteoporotic patients. This is, to our knowledge, the first report on peptide functionalization of RBCEVs and osteoclast-targeted delivery using RBCEVs.

A nano-targeted co-delivery system based on gene regulation and molecular blocking strategy for synergistic enhancement of platinum chemotherapy sensitivity in ovarian cancer

Ovarian cancer (OC) has a low five-year survival rate, mainly because of its drug resistance to chemotherapy. It is the key to reverse drug resistance to combine multiple sensitization pathways to play a synergistic role. A nano scaled targeted co-delivery system (P123-PEI-G12, PPG) modified by bifunctional peptide tLyP-1-NLS (G12) was fabricated by using Pluronic P123 conjugated with low molecular weight polyethyleneimine (PEI). This delivery system can co-delivery Olaparib (Ola) and p53 plasmids to synergistically enhance the sensitivity of OC to platinum-based chemotherapy. P53@P123-PEI-G2/Ola (Co-PPGs) can achieve efficient tumor accumulation and cellular internalization through G12-mediated targeting. Co-PPGs then break down in the tumor cells, releasing the drug. Co-PPGs significantly enhanced the sensitivity of cisplatin (DDP) in platinum-resistant ovarian cancer (PROC) and synergistically inhibited the proliferation of PROC in vitro and in vivo. The sensitizing and synergistic effects of Co-PPGs were related to the activation of p53, inhibition of poly-ADP-ribose polymerase (PARP) and p-glycoprotein (P-gp) expression. This work provides a promising strategy for the effective treatment of PROC.