Highly biased agonism for GPCR ligands via nanobody tethering.

Ligand-induced activation of G protein-coupled receptors (GPCRs) can initiate signaling through multiple distinct pathways with differing biological and physiological outcomes. There is intense interest in understanding how variation in GPCR ligand structure can be used to promote pathway selective signaling ("biased agonism") with the goal of promoting desirable responses and avoiding deleterious side effects. Here we present an approach in which a conventional peptide ligand for the type 1 parathyroid hormone receptor (PTHR1) is converted from an agonist which induces signaling through all relevant pathways to a compound that is highly selective for a single pathway. This is achieved not through variation in the core structure of the agonist, but rather by linking it to a nanobody tethering agent that binds with high affinity to a separate site on the receptor not involved in signal transduction. The resulting conjugate represents the most biased agonist of PTHR1 reported to date. This approach holds promise for facile generation of pathway selective ligands for other GPCRs.

Intestinal delivery of encapsulated bacteriocin peptides in cross-linked alginate microcapsules.

Oral delivery of larger bioactive peptides (>20 amino acids) to the small intestine remains a challenge due to their sensitivity to proteolytic degradation and chemical denaturation during gastrointestinal transit. In this study, we investigated the capacity of crosslinked alginate microcapsules (CLAMs) formed by spray drying to protect Plantaricin EF (PlnEF) (C-EF) in gastric conditions and to dissolve and release PlnEF in the small intestine. PlnEF is an unmodified, two-peptide (PlnE: 33 amino acids; PlnF: 34 amino acids) bacteriocin produced by Lactiplantibacillus plantarum with antimicrobial and gut barrier protective properties. After 2 h incubation in simulated gastric fluid (SGF) (pH 1.5), 43.39 % ± 8.27 % intact PlnEF was liberated from the CLAMs encapsulates, as determined by an antimicrobial activity assay. Transfer of the undissolved fraction to simulated intestinal fluid (SIF) (pH 7) for another 2 h incubation resulted in an additional release of 16.13 % ± 4.33 %. No active PlnEF was found during SGF or sequential SIF incubations when pepsin (2,000 U/ml) was added to the SGF. To test PlnEF release in C-EF contained in a food matrix, C-EF was mixed in peanut butter (PB) (0.15 g C-EF in 1.5 g PB). A total of 12.52 % ± 9.09 % active PlnEF was detected after incubation of PB + C-EF in SGF without pepsin, whereas no activity was found when pepsin was included. Transfer of the remaining PB + C-EF fractions to SIF yielded the recovery of 46.67 % ± 13.09 % and 39.42 % ± 11.53 % active PlnEF in the SIF following exposure to SGF and to SGF with pepsin, respectively. Upon accounting for the undissolved fraction after SIF incubation, PlnEF was fully protected in the CLAMs-PB mixture and there was not a significant reduction in active PlnEF when pepsin was present. These results show that CLAMs alone do not guard PlnEF bacteriocin peptides from gastric conditions, however, mixing them in PB protected against proteolysis and improved intestinal release.

Emergence of a short peptide based reductase via activation of the model hydride rich cofactor.

In extant biology, large and complex enzymes employ low molecular weight cofactors such as dihydronicotinamides as efficient hydride transfer agents and electron carriers for the regulation of critical metabolic processes. In absence of complex contemporary enzymes, these molecular cofactors are generally inefficient to facilitate any reactions on their own. Herein, we report short peptide-based amyloid nanotubes featuring exposed arrays of cationic and hydrophobic residues that can bind small molecular weak hydride transfer agents (NaBH4) to facilitate efficient reduction of ester substrates in water. In addition, the paracrystalline amyloid phases loaded with borohydrides demonstrate recyclability, substrate selectivity and controlled reduction and surpass the capabilities of standard reducing agent such as LiAlH4. The amyloid microphases and their collaboration with small molecular cofactors foreshadow the important roles that short peptide-based assemblies might have played in the emergence of protometabolism and biopolymer evolution in prebiotic earth.

A quick and innovative pipeline for producing chondrocyte-homing peptide-modified extracellular vesicles by three-dimensional dynamic culture of hADSCs spheroids to modulate the fate of remaining ear chondrocytes in the M1 macrophage-infiltrated microenvironment.

BACKGROUND: Extracellular vesicles (EVs) derived from human adipose-derived mesenchymal stem cells (hADSCs) have shown great therapeutic potential in plastic and reconstructive surgery. However, the limited production and functional molecule loading of EVs hinder their clinical translation. Traditional two-dimensional culture of hADSCs results in stemness loss and cellular senescence, which is unfavorable for the production and functional molecule loading of EVs. Recent advances in regenerative medicine advocate for the use of three-dimensional culture of hADSCs to produce EVs, as it more accurately simulates their physiological state. Moreover, the successful application of EVs in tissue engineering relies on the targeted delivery of EVs to cells within biomaterial scaffolds. METHODS AND RESULTS: The hADSCs spheroids and hADSCs gelatin methacrylate (GelMA) microspheres are utilized to produce three-dimensional cultured EVs, corresponding to hADSCs spheroids-EVs and hADSCs microspheres-EVs respectively. hADSCs spheroids-EVs demonstrate excellent production and functional molecule loading compared with hADSCs microspheres-EVs. The upregulation of eight miRNAs (i.e. hsa-miR-486-5p, hsa-miR-423-5p, hsa-miR-92a-3p, hsa-miR-122-5p, hsa-miR-223-3p, hsa-miR-320a, hsa-miR-126-3p, and hsa-miR-25-3p) and the downregulation of hsa-miR-146b-5p within hADSCs spheroids-EVs show the potential of improving the fate of remaining ear chondrocytes and promoting cartilage formation probably through integrated regulatory mechanisms. Additionally, a quick and innovative pipeline is developed for isolating chondrocyte homing peptide-modified EVs (CHP-EVs) from three-dimensional dynamic cultures of hADSCs spheroids. CHP-EVs are produced by genetically fusing a CHP at the N-terminus of the exosomal surface protein LAMP2B. The CHP + LAMP2B-transfected hADSCs spheroids were cultured with wave motion to promote the secretion of CHP-EVs. A harvesting method is used to enable the time-dependent collection of CHP-EVs. The pipeline is easy to set up and quick to use for the isolation of CHP-EVs. Compared with nontagged EVs, CHP-EVs penetrate the biomaterial scaffolds and specifically deliver the therapeutic miRNAs to the remaining ear chondrocytes. Functionally, CHP-EVs show a major effect on promoting cell proliferation, reducing cell apoptosis and enhancing cartilage formation in remaining ear chondrocytes in the M1 macrophage-infiltrated microenvironment. CONCLUSIONS: In summary, an innovative pipeline is developed to obtain CHP-EVs from three-dimensional dynamic culture of hADSCs spheroids. This pipeline can be customized to increase EVs production and functional molecule loading, which meets the requirements for regulating remaining ear chondrocyte fate in the M1 macrophage-infiltrated microenvironment.

A journey to your self: The vague definition of immune self and its practical implications.

The identification of immunogenic peptides has become essential in an increasing number of fields in immunology, ranging from tumor immunotherapy to vaccine development. The nature of the adaptive immune response is shaped by the similarity between foreign and self-protein sequences, a concept extensively applied in numerous studies. Can we precisely define the degree of similarity to self? Furthermore, do we accurately define immune self? In the current work, we aim to unravel the conceptual and mechanistic vagueness hindering the assessment of self-similarity. Accordingly, we demonstrate the remarkably low consistency among commonly employed measures and highlight potential avenues for future research.

So Let's Design a Peptide . . . A Physician's Approach.

This paper outlines a process undertaken by a physician to design a peptide aimed at impacting the extracellular matrix. From a position of very little expertise, a new peptide was designed with amino acid constituents based on the structural proteins collagen and elastin. Sequencing was also considered, given the periodic repetition observed in these proteins, and a peptide with reasonable molecular weight and physical characteristics was designed using available software. The sequence of events concerning intellectual property, functionality investigation, and eventual use of the peptide in new formulations is detailed. This may be of interest to physicians who consider this exercise out of the scope of the usual practice. J Drugs Dermatol. 2024;23(5):347-352.    doi:10.36849/JDD.7921.

Stress Granule Core Protein-Derived Peptides Inhibit Assembly of Stress Granules and Improve Sorafenib Sensitivity in Cancer Cells.

Upon a variety of environmental stresses, eukaryotic cells usually recruit translational stalled mRNAs and RNA-binding proteins to form cytoplasmic condensates known as stress granules (SGs), which minimize stress-induced damage and promote stress adaptation and cell survival. SGs are hijacked by cancer cells to promote cell survival and are consequently involved in the development of anticancer drug resistance. However, the design and application of chemical compounds targeting SGs to improve anticancer drug efficacy have rarely been studied. Here, we developed two types of SG inhibitory peptides (SIPs) derived from SG core proteins Caprin1 and USP10 and fused with cell-penetrating peptides to generate TAT-SIP-C1/2 and SIP-U1-Antp, respectively. We obtained 11 SG-inducing anticancer compounds from cell-based screens and explored the potential application of SIPs in overcoming resistance to the SG-inducing anticancer drug sorafenib. We found that SIPs increased the sensitivity of HeLa cells to sorafenib via the disruption of SGs. Therefore, anticancer drugs which are competent to induce SGs could be combined with SIPs to sensitize cancer cells, which might provide a novel therapeutic strategy to alleviate anticancer drug resistance.

Copper(I)-nitrene platform for chemoproteomic profiling of methionine.

Methionine plays a critical role in various biological and cell regulatory processes, making its chemoproteomic profiling indispensable for exploring its functions and potential in protein therapeutics. Building on the principle of rapid oxidation of methionine, we report Copper(I)-Nitrene Platform for robust, and selective labeling of methionine to generate stable sulfonyl sulfimide conjugates under physiological conditions. We demonstrate the versatility of this platform to label methionine in bioactive peptides, intact proteins (6.5-79.5 kDa), and proteins in complex cell lysate mixtures with varying payloads. We discover ligandable proteins and sites harboring hyperreactive methionine within the human proteome. Furthermore, this has been utilized to profile oxidation-sensitive methionine residues, which might increase our understanding of the protective role of methionine in diseases associated with elevated levels of reactive oxygen species. The Copper(I)-Nitrene Platform allows labeling methionine residues in live cancer cells, observing minimal cytotoxic effects and achieving dose-dependent labeling. Confocal imaging further reveals the spatial distribution of modified proteins within the cell membrane, cytoplasm, and nucleus, underscoring the platform's potential in profiling the cellular interactome.

DeepAVP-TPPred: identification of antiviral peptides using transformed image-based localized descriptors and binary tree growth algorithm.

MOTIVATION: Despite the extensive manufacturing of antiviral drugs and vaccination, viral infections continue to be a major human ailment. Antiviral peptides (AVPs) have emerged as potential candidates in the pursuit of novel antiviral drugs. These peptides show vigorous antiviral activity against a diverse range of viruses by targeting different phases of the viral life cycle. Therefore, the accurate prediction of AVPs is an essential yet challenging task. Lately, many machine learning-based approaches have developed for this purpose; however, their limited capabilities in terms of feature engineering, accuracy, and generalization make these methods restricted. RESULTS: In the present study, we aim to develop an efficient machine learning-based approach for the identification of AVPs, referred to as DeepAVP-TPPred, to address the aforementioned problems. First, we extract two new transformed feature sets using our designed image-based feature extraction algorithms and integrate them with an evolutionary information-based feature. Next, these feature sets were optimized using a novel feature selection approach called binary tree growth Algorithm. Finally, the optimal feature space from the training dataset was fed to the deep neural network to build the final classification model. The proposed model DeepAVP-TPPred was tested using stringent 5-fold cross-validation and two independent dataset testing methods, which achieved the maximum performance and showed enhanced efficiency over existing predictors in terms of both accuracy and generalization capabilities. AVAILABILITY AND IMPLEMENTATION: https://github.com/MateeullahKhan/DeepAVP-TPPred.

Recent Advances in the Field of Amino Acid-Conjugated Aminoferrocenes-A Personal Perspective.

The development of turn-based inhibitors of protein-protein interactions has attracted considerable attention in medicinal chemistry. Our group has synthesized a series of peptides derived from an amino-functionalized ferrocene to investigate their potential to mimic protein turn structures. Detailed DFT and spectroscopic studies (IR, NMR, CD) have shown that, for peptides, the backbone chirality and bulkiness of the amino acid side chains determine the hydrogen-bond pattern, allowing tuning of the size of the preferred hydrogen-bonded ring in turn-folded structures. However, their biological potential is more dependent on their lipophilicity. In addition, our pioneering work on the chiroptical properties of aminoferrocene-containing peptides enables the correlation of their geometry with the sign of the CD signal in the absorption region of the ferrocene chromophore. These studies have opened up the possibility of using aminoferrocene and its derivatives as chirooptical probes for the determination of various chirality elements, such as the central chirality of amino acids and the helicity of peptide sequences.

Tumor-Derived Antigenic Peptides as Potential Cancer Vaccines.

Peptide antigens derived from tumors have been observed to elicit protective immune responses, categorized as either tumor-associated antigens (TAAs) or tumor-specific antigens (TSAs). Subunit cancer vaccines incorporating these antigens have shown promise in inducing protective immune responses, leading to cancer prevention or eradication. Over recent years, peptide-based cancer vaccines have gained popularity as a treatment modality and are often combined with other forms of cancer therapy. Several clinical trials have explored the safety and efficacy of peptide-based cancer vaccines, with promising outcomes. Advancements in techniques such as whole-exome sequencing, next-generation sequencing, and in silico methods have facilitated the identification of antigens, making it increasingly feasible. Furthermore, the development of novel delivery methods and a deeper understanding of tumor immune evasion mechanisms have heightened the interest in these vaccines among researchers. This article provides an overview of novel insights regarding advancements in the field of peptide-based vaccines as a promising therapeutic avenue for cancer treatment. It summarizes existing computational methods for tumor neoantigen prediction, ongoing clinical trials involving peptide-based cancer vaccines, and recent studies on human vaccination experiments.

On the Re-Creation of Protoribosome Analogues in the Lab.

The evolution of the translation system is a fundamental issue in the quest for the origin of life. A feasible evolutionary scenario necessitates the autonomous emergence of a protoribosome capable of catalyzing the synthesis of the initial peptides. The peptidyl transferase center (PTC) region in the modern ribosomal large subunit is believed to retain a vestige of such a prebiotic non-coded protoribosome, which would have self-assembled from random RNA chains, catalyzed peptide bond formation between arbitrary amino acids, and produced short peptides. Recently, three research groups experimentally demonstrated that several distinct dimeric constructs of protoribosome analogues, derived predicated on the approximate 2-fold rotational symmetry inherent in the PTC region, possess the ability to spontaneously fold, dimerize, and catalyze the formation of peptide bonds and of short peptides. These dimers are examined, aiming at retrieving information concerned with the characteristics of a prebiotic protoribosome. The analysis suggests preconditions for the laboratory re-creation of credible protoribosome analogues, including the preference of a heterodimer protoribosome, contradicting the common belief in the precedence of homodimers. Additionally, it derives a dynamic process which possibly played a role in the spontaneous production of the first bio-catalyzed peptides in the prebiotic world.

The First Reciprocal Activities of Chiral Peptide Pharmaceuticals: Thymogen and Thymodepressin, as Examples.

Peptides show high promise in the targeting and intracellular delivery of next-generation biotherapeutics. The main limitation is peptides' susceptibility to proteolysis in biological systems. Numerous strategies have been developed to overcome this challenge by chemically enhancing the resistance to proteolysis. In nature, amino acids, except glycine, are found in L- and D-enantiomers. The change from one form to the other will change the primary structure of polypeptides and proteins and may affect their function and biological activity. Given the inherent chiral nature of biological systems and their high enantiomeric selectivity, there is rising interest in manipulating the chirality of polypeptides to enhance their biomolecular interactions. In this review, we discuss the first examples of up-and-down homeostasis regulation by two enantiomeric drugs: immunostimulant Thymogen (L-Glu-L-Trp) and immunosuppressor Thymodepressin (D-Glu(D-Trp)). This study shows the perspective of exploring chirality to remove the chiral wall between L- and D-biomolecules. The selected clinical result will be discussed.

A Coronin 1-Derived Peptide Inhibits Membrane Fusion by Modulating Membrane Organization and Dynamics.

Membrane fusion is considered the first step in the entry of enveloped viruses into the host cell. Several targeted strategies have been implemented to block viral entry by limiting the fusion protein to form a six-helix bundle, which is a prerequisite for fusion. Nonetheless, the development of broad-spectrum fusion inhibitors is essential to combat emerging and re-emerging viral infections. TG-23, a coronin 1, a tryptophan-aspartate-rich phagosomal protein-derived peptide, demonstrated inhibition of fusion between small unilamellar vesicles (SUVs) by modulating the membrane's physical properties. However, its inhibitory efficacy reduces with an increasing concentration of membrane cholesterol. The present work aims to develop a fusion inhibitor whose efficacy would be unaltered in the presence of membrane cholesterol. A stretch of the tryptophan-aspartic acid-containing peptide with a similar secondary structure and hydrophobicity profile of TG-23 from coronin 1 was synthesized, and its ability to inhibit SUV-SUV fusion with varying concentrations of membrane cholesterol was evaluated. Our results demonstrate that the GG-21 peptide inhibits fusion irrespective of the cholesterol content of the membrane. We have further evaluated the peptide-induced change in the membrane organization and dynamics utilizing arrays of steady-state and time-resolved fluorescence measurements and correlated these results with their effect on fusion. Interestingly, GG-21 displays inhibitory efficacy in a wide variety of lipid compositions despite having a secondary structure and physical properties similar to those of TG-23. Overall, our results advocate that the secondary structure and physical properties of the peptide may not be sufficient to predict its inhibitory efficacy.

Innovative peptide architectures: advancements in foldamers and stapled peptides for drug discovery.

INTRODUCTION: Peptide foldamers play a critical role in pharmaceutical research and biomedical applications. This review highlights recent (post-2020) advancements in novel foldamers, synthetic techniques, and their applications in pharmaceutical research. AREAS COVERED: The authors summarize the structures and applications of peptide foldamers such as α, ß, γ-peptides, hydrocarbon-stapled peptides, urea-type foldamers, sulfonic-γ-amino acid foldamers, aromatic foldamers, and peptoids, which tackle the challenges of traditional peptide drugs. Regarding antimicrobial use, foldamers have shown progress in their potential against drug-resistant bacteria. In drug development, peptide foldamers have been used as drug delivery systems (DDS) and protein-protein interaction (PPI) inhibitors. EXPERT OPINION: These structures exhibit resistance to enzymatic degradation, are promising for therapeutic delivery, and disrupt crucial PPIs associated with diseases such as cancer with specificity, versatility, and stability, which are useful therapeutic properties. However, the complexity and cost of their synthesis, along with the necessity for thorough safety and efficacy assessments, necessitate extensive research and cross-sector collaboration. Advances in synthesis methods, computational modeling, and targeted delivery systems are essential for fully realizing the therapeutic potential of foldamers and integrating them into mainstream medical treatments.

Insights into modifiers effects in differential mobility spectrometry: A data science approach for metabolomics and peptidomics.

Utilizing a data-driven approach, this study investigates modifier effects on compensation voltage in differential mobility spectrometry-mass spectrometry (DMS-MS) for metabolites and peptides. Our analysis uncovers specific factors causing signal suppression in small molecules and pinpoints both signal suppression mechanisms and the analytes involved. In peptides, machine learning models discern a relationship between molecular weight, topological polar surface area, peptide charge, and proton transfer-induced signal suppression. The models exhibit robust performance, offering valuable insights for the application of DMS to metabolites and tryptic peptides analysis by DMS-MS.

Two Novel Angiotensin-Converting Enzyme (ACE) Inhibitory and ACE2 Upregulating Peptides from the Hydrolysate of Pumpkin (Cucurbita moschata) Seed Meal.

Pumpkin (Cucurbita moschata) seed meal (PSM), the major byproduct of pumpkin seed oil industry, was used to prepare angiotensin-converting enzyme (ACE) inhibitory and angiotensin-converting enzyme 2 (ACE2) upregulating peptides. These peptides were isolated and purified from the PSM hydrolysate prepared using Neutrase 5.0 BG by ultrafiltration, Sephadex G-15 column chromatography, and reversed-phase high-performance liquid chromatography. Two peptides with significant ACE inhibition activity were identified as SNHANQLDFHP and PVQVLASAYR with IC50 values of 172.07 and 90.69 µM, respectively. The C-terminal tripeptides of the two peptides contained Pro, Phe, and Tyr, respectively, and PVQVLASAYR also had Val in its N-terminal tripeptide, which was a favorable structure for ACE inhibition. Molecular docking results declared that the two peptides could interact with ACE through hydrogen bonds and hydrophobic interactions. Furthermore, the two peptides performed protective function on EA.hy926 cells by decreasing the secretion of endothelin-1, increasing the release of nitric oxide, and regulating the ACE2 activity. In vitro simulated gastrointestinal digestion showed the two peptides exhibited good stability against gastrointestinal enzyme digestion. In conclusion, PSM is a promising material for preparing antihypertensive peptides.

Fabrication of an Antifouling Surface Plasmon Resonance Sensor with Stratified Zwitterionic Peptides for Highly Efficient Detection of Peanut Allergens in Biscuits.

Peanut allergen monitoring is currently an effective strategy to avoid allergic diseases, while food matrix interference is a critical challenge during detection. Here, we developed an antifouling surface plasmon resonance sensor (SPR) with stratified zwitterionic peptides, which provides both excellent antifouling and sensing properties. The antifouling performance was measured by the SPR, which showed that stratified peptide coatings showed much better protein resistance, reaching ultralow adsorption levels (<5 ng/cm2). Atomic force microscopy was used to further analyze the antifouling mechanism from a mechanical perspective, which demonstrated lower adsorption forces on hybrid peptide coatings, confirming the better antifouling performance of stratified surfaces. Moreover, the recognition of peanut allergens in biscuits was performed using an SPR with high efficiency and appropriate recovery results (98.2-112%), which verified the feasibility of this assay. Therefore, the fabrication of antifouling sensors with stratified zwitterionic peptides provides an efficient strategy for food safety inspection.

Affinity-Driven Aryl Diazonium Labeling of Peptide Receptors on Living Cells.

Peptide-receptor interactions play critical roles in a wide variety of physiological processes. Methods to link bioactive peptides covalently to unmodified receptors on the surfaces of living cells are valuable for studying receptor signaling, dynamics, and trafficking and for identifying novel peptide-receptor interactions. Here, we utilize peptide analogues bearing deactivated aryl diazonium groups for the affinity-driven labeling of unmodified receptors. We demonstrate that aryl diazonium-bearing peptide analogues can covalently label receptors on the surface of living cells using both the neurotensin and the glucagon-like peptide 1 receptor systems. Receptor labeling occurs in the complex environment of the cell surface in a sequence-specific manner. We further demonstrate the utility of this covalent labeling approach for the visualization of peptide receptors by confocal fluorescence microscopy and for the enrichment and identification of labeled receptors by mass spectrometry-based proteomics. Aryl diazonium-based affinity-driven receptor labeling is attractive due to the high abundance of tyrosine and histidine residues susceptible to azo coupling in the peptide binding sites of receptors, the ease of incorporation of aryl diazonium groups into peptides, and the relatively small size of the aryl diazonium group. This approach should prove to be a powerful and relatively general method to study peptide-receptor interactions in cellular contexts.

Oral Synergism of Egg-White-Derived Peptides (EWDP) and Curcumin for Colitis Mitigation via Polysaccharide/Cyclodextrin Metal-Organic Framework-Based Assemblies.

Recently, oral deliverable strategies of multiple nutraceuticals for ulcerative colitis (UC) mitigation have attracted increasing attention. This study aimed to fabricate facile oral assemblies loaded with egg-white-derived peptides (EWDP) and curcumin based on carboxymethyl chitosan (CMCS) and an γ-cyclodextrin metal-organic framework (MOF). Herein, outer CMCS could coassemble with EWDP (both nutraceuticals and building blocks) into cobweb-like fibrils to promote bridging with inner MOF via coordinative noncovalent interactions (hydrogen bonding, hydrophobic interaction, and electrostatic interaction). Compared with conventional γ-cyclodextrin/MOF-based composites, the above coassembly could also endow the biocompatible assemblies with superior nanoscale colloidal properties, processing applicability (curcumin storage stability, bioaccessibility, and aqueous solubility), and bioactivity. Moreover, the oral synergism of EWDP and curcumin (initially nonsynergistic) for UC mitigation was achieved by alleviating inflammatory damage and gut microbiota imbalance. Overall, the novel assemblies could be a promising amplifier and platform to facilitate oral formulations of various nutraceuticals for food processing and UC relief.