Peptide Block Research Articles

Molecular Insights into the Self-Assembly of Block Copolymer Suckerin Polypeptides into Nanoconfined β-Sheets.

Suckerin in squid sucker ring teeth is a block-copolymer peptide comprised of two repeating modules-the alanine and histidine-rich M1 and the glycine-rich M2. Suckerin self-assemblies display excellent thermo-plasticity and pH-responsive properties, along with the high biocompatibility, biodegradability, and sustainability. However, the self-assembly mechanism and the detailed role of each module are still elusive, limiting the capability of applying and manipulating such biomaterials. Here, the self-assembly dynamics of the two modules and two minimalist suckerin-mimetic block-copolymers, M1-M2-M1 and M2-M1-M2, in silico is investigated. The simulation results demonstrate that M2 has a stronger self-association but weaker β-sheet propensities than M1. The high self-assembly propensity of M2 allows the minimalist block-copolymer peptides to coalesce with microphase separation, enabling the formation of nanoconfined β-sheets in the matrix formed by M1-M2 contacts. Since these glycine-rich fragments with scatted hydrophobic and aromatic residues are building blocks of many other block-copolymer peptides, the study suggests that these modules function as the "molecular glue" in addition to the flexible linker or spacer to drive the self-assembly and microphase separation. The uncovered molecular insights may help understand the structure and function of suckerin and also aid in the design of functional block-copolymer peptides for nanotechnology and biomedicine applications.

A Density Functional Theory and Information-Theoretic Approach Study of Interaction Energy and Polarizability for Base Pairs and Peptides.

Using density functional theory (DFT) and the information-theoretic approach (ITA) quantities to appreciate the energetics and properties of biopolymers is still an unaccomplished and ongoing task. To this end, we studied the building blocks of nucleic acid base pairs and small peptides. For base pairs, we have dissected the relative importance of energetic components by using two energy partition schemes in DFT. Our results convincingly show that the exchange-correlation effect predominantly governs the molecular stability of base pairs while the electrostatic potential plays a minor but indispensable role, and the steric effect is trivial. Furthermore, we have revealed that simple density-based ITA functions are in good relationships with molecular polarizabilities for a series of 30 hydrogen-bonded base pairs and all 20 natural α-amino acids, 400 dipeptides, and 8000 tripeptides. Based on these lines, one can easily predict the molecular polarizabilities of larger peptides, even proteins as long as the total molecular wavefunction is available, rather than solving the computationally demanding coupled-perturbed Hartree–Fock (CPHF) equation or its DFT counterpart coupled-perturbed Kohn–Sham (CPKS) equation.

A Novel CaMKII Inhibitory Peptide Blocks Relapse to Morphine Seeking by Influencing Synaptic Plasticity in the Nucleus Accumbens Shell.

Drugs of abuse cause enduring functional disorders in the brain reward circuits, leading to cravings and compulsive behavior. Although people may rehabilitate by detoxification, there is a high risk of relapse. Therefore, it is crucial to illuminate the mechanisms of relapse and explore the therapeutic strategies for prevention. In this research, by using an animal model of morphine self-administration in rats and a whole-cell patch–clamp in brain slices, we found changes in synaptic plasticity in the nucleus accumbens (NAc) shell were involved in the relapse to morphine-seeking behavior. Compared to the controls, the amplitude of long-term depression (LTD) induced in the medium spiny neurons increased after morphine self-administration was established, recovered after the behavior was extinguished, and increased again during the relapse induced by morphine priming. Intravenous injection of MA, a new peptide obtained by modifying Ca2+/calmodulin-dependent protein kinase II (CaMKII) inhibitor “myr-AIP”, decreased CaMKII activity in the NAc shell and blocked the reinstatement of morphine-seeking behavior without influence on the locomotor activity. Moreover, LTD was absent in the NAc shell of the MA-pretreated rats, whereas it was robust in the saline controls in which morphine-seeking behavior was reinstated. These results indicate that CaMKII regulates morphine-seeking behavior through its involvement in the change of synaptic plasticity in the NAc shell during the relapse, and MA may be of great value in the clinical treatment of relapse to opioid seeking.

Controlling self-assembly of co-polypeptide by block ratio and block sequence

Block ratio and block sequence of polypeptide influenced the self-assembly of copolymer significantly. Tuning self-assembly to form various nanostructures with different properties through molecular design was a rather feasible strategy. Triblock copolymers based on poly( l -alanine) (PA), poly( l -valine) (PV) and poly (ethylene glycol) (PEG) with various block ratio and block sequence were synthesized. The number of amino acid residues was kept at 10. The sol-gel transition behavior, self-assemble nanostructure, secondary structure of the copolymers in aqueous solution were investigated to reveal that PA block between PEG and PV segment interfered the β-sheet structure, resulting in the reduced or increased sol-gel transition temperature or precipitation depended on the block ratio of PA and PV. Shorter PA or PV block displayed more significant influence on sol-gel transition. Interestingly, PV block between PEG and PA segment induced the formation of more stable β-sheet stacking structures, leading to sol-gel transition at higher temperature. PEG dehydration and the enhancement of ordered structure with temperature rising caused the sol-gel transition of these copolymers. We concluded herein that disordered structure in co-polypeptide can enhance thermo-sensitivity. • Conformation of synthesized non-natural polypeptides based on side-chain design. • Block sequence of non-natural polypeptides affected sol-gel transition. • Shorter peptide block influenced more significantly on sol-gel transition. • Less hydrophobic and weaker steric hindrance block at the tail enhanced the stability of nanostructures. • Disordered structure in co-polypeptide enhanced thermo-sensitivity.

Deciphering the conformational landscape of few selected aromatic noncoded amino acids (NCAAs) for applications in rational design of peptide therapeutics

Amino acids are the essential building blocks of both synthetic and natural peptides, which are crucial for biological functions and also important as biological probes for mapping the complex protein–protein interactions (PPIs) in both prokaryotic and eukaryotic systems. Mapping the PPIs through the chemical biology approach provides pharmacologically relevant peptides, which can have agonistic or antagonistic effects on the targeted biological systems. It is evidenced that ≥ 60 peptide-based drugs have been approved by the US-FDA so far, and the number will improve further in the foreseeable future, as ≥ 140 peptides are currently in clinical trials. However, natural peptides often require fine-tuning of their pharmacological properties by strategically replacing the αL-amino acids of the peptides with non-coded amino acids (NCAA), for which codons are absent in the genetic code for biosynthesis of proteins, prior to their applications as therapeutics. Considering the diverse repertoire of the NCAAs, the conformational space of many NCAAs is yet to be explored systematically in the context of the rational design of therapeutic peptides. The current study deciphers the conformational landscape of a few such Cα-substituted aromatic NCAAs (Ing: 2-indanyl-l-Glycine; Bpa: 4-benzoyl-l-phenylalanine; Aic: 2-aminoindane-2-carboxylic acid) both in the context of tripeptides and model synthetic peptide sequences, using alanine (Ala) and proline (Pro) as the reference. The combined data obtained from the computational and biophysical studies indicate the general success of this approach, which can be exploited further to rationally design optimized peptide sequences of unusual architecture with potent antimicrobial, antiviral, gluco-regulatory, immunomodulatory, and anti-inflammatory activities. Supplementary InformationThe online version contains supplementary material available at 10.1007/s00726-022-03175-z.

Star-Shaped Peptide-Polymer Hybrids as Fast pH-Responsive Supramolecular Hydrogels.

Significant challenges have gone into the design of smart hydrogels, with numerous potential applications in the industrial, cosmetic, and biomedical fields. Herein, we report the synthesis of novel 4-arm self-assembling peptide-polyethylene glycol (PEG) hybrid star-shaped polymers and their comprehensive hydrogel properties. β-sheet-forming oligopeptides with alternating hydrophobic Leu/ionizable Glu repeats and Cys residues were successfully conjugated to 4-arm PEG via a thiol-maleimide click reaction. The hybrid star-shaped polymers demonstrated good cytocompatibility and reversible β-sheet (lightly acidic pH)-to-random coil (neutral and basic pH) transition in dilute aqueous solutions. At increasing polymer concentrations up to 0.5 wt %, the star-shaped polymers formed transparent hydrogels with shear-thinning and self-healing behaviors via β-sheet self-assembly, as well as a conformation-dependent gel-sol transition. Interestingly, the star-shaped polymers responded rapidly to pH changes, causing gelation to occur rapidly within a few seconds from the change in pH. Hydrogel characteristics could be modulated by manipulating the length and net charge of the peptide blocks. Furthermore, these star-shaped polymers served as satisfactory network scaffolds that could respond to dynamic environmental changes in the pH-oscillation system, owing to their excellent gelation capability and pH sensitivity. As such, they are highly favorable for diverse applications, such as pH-responsive controlled release.

Abstract 3599: Helical stapled peptides derived from DIRAS3 block KRAS dimerization and downstream MEK/ERK signaling in pancreatic and ovarian carcinomas

Abstract The overall 5-year survival for pancreatic ductal adenocarcinoma (PDAC) has changed little over the past few decades, and PDAC is predicted to become the second leading cause of cancer-related mortality in the next decade in Western countries. Low grade serous ovarian cancer (LGSOC) is a slow-growing but generally chemo-resistant cancer with few effective treatments for recurrent disease. More than 90% of PDAC and up to 40% of LGSOC express mutationally activated KRAS that drives persistent cell division, anti-apoptosis, cell migration and metastasis. Preventing signaling from the KRAS oncoprotein has been challenging. While recent studies have shown promising results by targeting the binding pocket in Switch II for development of KRAS mutant-specific inhibitors for the G12C mutant and G12D mutant, targeting other mutations remains a work in progress. We have discovered that DIRAS3, a novel endogenous physiological RAS inhibitor, blocks KRAS activity by directly binding KRAS with high affinity, inhibiting KRAS dimerization/nanoclustering and blocking effector activation. In this study we have developed drug-like, helical 10-mer stapled peptides derived from DIRAS3 α5 domain. Preliminary studies show that these lead compounds associate with KRAS with low nanomolar affinity, are largely resistant to serum proteases, and rapidly cross the cellular membrane. Functionally, DIRAS3 peptides—but not control peptides—block KRAS homodimers in ReBiL split luciferase assays of KRAS dimerization and disrupt nanoclustering by TEM analysis of GFP-KRAS(G12V)-labeled gold nanoparticles on the inner leaflet of the plasma membrane of cells. Moreover, DIRAS3 peptides significantly inhibit phospho-ERK, decrease cell viability and induce apoptosis in pancreatic and low-grade ovarian cancer cells with KRAS mutations. Finally, daily treatment with stapled DIRAS3-derived peptides inhibited growth of ASPC-1 (KRAS G12D) PDAC xenografts and improved survival. Thus, our study suggests that development of DIRAS3 stapled peptides may provide a novel therapeutic approach to target mutant KRAS-driven cancers. Citation Format: Joshua P. Gray, Gamze E. Bildik, Margie N. Sutton, Yong Zhou, Steven Millward, John F. Hancock, Zhen Lu, Robert C. Bast. Helical stapled peptides derived from DIRAS3 block KRAS dimerization and downstream MEK/ERK signaling in pancreatic and ovarian carcinomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3599.

Recombinant Integrin β1 Signal Peptide Blocks Gliosis Induced by Aβ Oligomers.

Glial cells participate actively in the early cognitive decline in Alzheimer’s disease (AD) pathology. In fact, recent studies have found molecular and functional abnormalities in astrocytes and microglia in both animal models and brains of patients suffering from this pathology. In this regard, reactive gliosis intimately associated with amyloid plaques has become a pathological hallmark of AD. A recent study from our laboratory reports that astrocyte reactivity is caused by a direct interaction between amyloid beta (Aβ) oligomers and integrin β1. Here, we have generated four recombinant peptides including the extracellular domain of integrin β1, and evaluated their capacity both to bind in vitro to Aβ oligomers and to prevent in vivo Aβ oligomer-induced gliosis and endoplasmic reticulum stress. We have identified the minimal region of integrin β1 that binds to Aβ oligomers. This region is called signal peptide and corresponds to the first 20 amino acids of the integrin β1 N-terminal domain. This recombinant integrin β1 signal peptide prevented Aβ oligomer-induced ROS generation in primary astrocyte cultures. Furthermore, we carried out intrahippocampal injection in adult mice of recombinant integrin β1 signal peptide combined with or without Aβ oligomers and we evaluated by immunohistochemistry both astrogliosis and microgliosis as well as endoplasmic reticulum stress. The results show that recombinant integrin β1 signal peptide precluded both astrogliosis and microgliosis and endoplasmic reticulum stress mediated by Aβ oligomers in vivo. We have developed a molecular tool that blocks the activation of the molecular cascade that mediates gliosis via Aβ oligomer/integrin β1 signaling.

Preparation of Non-Toxic Fluorescent Peptide-Coated Silica/PEG Nanoparticles from Peptide-Block Copolymer Conjugates

Peptide-decorated nanosystems have demonstrated higher stability and improved cellular uptake as compared to bare NPs and appear highly promising in diagnostics and theranostics of cancer. Herein, we discuss the preparation and structural characterization of peptide-functionalized silica/PEG NPs, starting from peptide–block copolymers, prepared in turn by conjugation of the peptides to block copolymers before NP formation. This synthetic design allowed full control of density and composition of peptide surface coverage. Preliminary experiments support the low toxicity of the fluorescent peptide–NPs and their ability of cell internalization.

Genomics-Based Reconstruction and Predictive Profiling of Amino Acid Biosynthesis in the Human Gut Microbiome.

The human gut microbiota (HGM) have an impact on host health and disease. Amino acids are building blocks of proteins and peptides, also serving as precursors of many essential metabolites including nucleotides, cofactors, etc. Many HGM community members are unable to synthesize some amino acids (auxotrophs), while other members possess complete biosynthetic pathways for these nutrients (prototrophs). Metabolite exchange between auxotrophs and prototrophs affects microbial community structure. Previous studies of amino acid biosynthetic phenotypes were limited to model species or narrow taxonomic groups of bacteria. We analyzed over 2800 genomes representing 823 cultured HGM species with the aim to reconstruct biosynthetic pathways for proteinogenic amino acids. The genome context analysis of incomplete pathway variants allowed us to identify new potential enzyme variants in amino acid biosynthetic pathways. We further classified the studied organisms with respect to their pathway variants and inferred their prototrophic vs. auxotrophic phenotypes. A cross-species comparison was applied to assess the extent of conservation of the assigned phenotypes at distinct taxonomic levels. The obtained reference collection of binary metabolic phenotypes was used for predictive metabolic profiling of HGM samples from several large metagenomic datasets. The established approach for metabolic phenotype profiling will be useful for prediction of overall metabolic properties, interactions, and responses of HGM microbiomes as a function of dietary variations, dysbiosis and other perturbations.

Vasoactive Intestinal Peptide Amphiphile Micelle Chemical Structure and Hydrophobic Domain Influence Immunomodulatory Potentiation.

Vasoactive intestinal peptide (VIP) is a neuropeptide capable of downregulating innate immune responses in antigen presenting cells (APCs) by suppressing their pro-inflammatory cytokine secretion and cell surface marker expression. Though VIP's bioactivity could possibly be leveraged as a treatment for transplant tolerance, drug delivery innovation is required to overcome its intrinsically limited cellular delivery capacity. One option is to employ peptide amphiphiles (PAs) which are lipidated peptides capable of self-assembling into micelles in water that can enhance cellular association. With this approach in mind, a series of triblock VIP amphiphiles (VIPAs) has been synthesized to explore the influence of block arrangement and hydrophobicity on micelle biocompatibility and bioactivity. VIPA formulation has been found to influence the shape, size, and surface charge of VIPA micelles (VIPAMs) as well as their cytotoxicity and immunomodulatory effects. Specifically, the enclosed work provides strong evidence that cylindrical VIPAMs with aspect ratios of 1.5-150 and moderate positive surface charge are able to potentiate the bioactivity of VIP limiting TNF-α secretion and MHC II and CD86 surface expression on APCs. With these criteria, we have identified PalmK-(EK)4-VIP as our lead formulation, which showed comparable or enhanced anti-inflammatory effects relative to the unmodified VIP at all dosages evaluated. Additionally, the relationships between peptide block location and lipid block size provide further information on the chemical structure-function relationships of PA micelles for the delivery of VIP as well as potentially for other peptides more broadly.

Structure-function relationship of small peptides generated during the ripening of Spanish dry-cured ham: Peptidome, molecular stability and computational modelling

A systematic insight into the structure–function properties of small bioactive peptides is of great importance. Herein, peptidomics and computational methodology were adopted to investigate the stabilization patterns and building blocks of antioxidant peptides resulting from proteolysis during the ripening of Spanish dry-cured ham (9–24 months of processing). The results showed that native peptides underwent manufacture-induced steric/redox stress, while homogeneous/heterogeneous p-π/π-π interaction significantly improved the ABTS+ inhibition activity of hydrophobic peptides. However, for more hydrophilic peptides, the intrinsic π-interaction system (i.e., cation-π and π-π packing) substantially interfered with ABTS+/DPPH scavenging events when compared to the aromatic residues. Semi-quantitative peptidomics and molecular simulation/docking revealed that VFSSQGQSELILLQK and LCPSPDGLYL are two potential antioxidant peptides at the late ripening-drying. They had distinctive self-folding destinies following solvation owing to varying charged/hydrophobic properties of termini and hydrogen atom donor, allowing different flexibility of backbone and interactive surface towards free radicals ex situ followed by electron/proton transfer.

Peptide Blocking Self-Polymerization of Extracellular Calcium-Sensing Receptor Attenuates Hypoxia-Induced Pulmonary Hypertension.

[Figure: see text].

Atroposelective Dynamic Kinetic Resolution via In Situ Hemiaminals Catalyzed by N-Heterocyclic Carbene.

Axially chiral amino acids and its derivatives are vital building blocks of bioactive molecules, artificial peptides, and asymmetric catalysts. Herein, we report an unprecedented carbene-catalyzed atroposelective dynamic kinetic resolution to access axially chiral amino esters via in situ hemiaminals. This protocol features a broad substrate scope and good functional group tolerance and allows the rapid assembly of axially chiral amino esters in good to high yields with high enantioselectivities.

A facile approach towards amidinophenylalanine derivatives as building blocks for the synthesis of non-natural peptides and peptidomimetics

The amidine function is extensively utilized as synthetic intermediate for structurally diverse heterocycles and as a core element in drugs and drug candidates. This study describes a facile and convenient approach for the highly efficient synthesis of amidinophenylalanine derivatives via the reduction of amidoximophenylalanines in the presence of Ac2O/AcOH/Zn at room temperature. The described process is attractive for process and medicinal chemistry due to its facility, safety, low cost, applicability for hydrogenation-sensitive compounds, and efficiency. Using this method, amidinophenylalanine derivatives as building blocks for peptides were obtained in excellent yields.

Nanocapsules Produced by Nanoprecipitation of Designed Suckerin-Silk Fusion Proteins

Herein, we report on the precise design of a modular fusion protein amenable to the construction of nanocapsules by nanoprecipitation. The central squid suckerin-derived peptide block provides structural stability, whereas both termini from spider silk fibroins make the protein highly soluble at physiological pH, a critical requirement for the nanoprecipitation process. With this design, nanocapsules consisting of fusion protein shells and oily cores with sizes in the range of 190-250 nm are built in a straightforward manner.

Poly(Ethylene Glycol)-Poly(l-Alanine)/Hyaluronic Acid Complex as a 3D Platform for Understanding Cancer Cell Migration in the Tumor Microenvironment.

Cancer progression and migration in the tumor microenvironment are related to cell types and three-dimensional (3D) matrices. Therefore, developing biomimetic tumor models, including co-culture systems and a tunable 3D matrix, could play an essential role in understanding the cancer environment. Here, multicellular spheroids using human adipose-derived mesenchymal stem cells (hADSCs) and breast cancer cells (MDA-MB-231) within the 3D matrix were used as a tumor microenvironment (TME) mimicking platform. The amphiphilic peptide block copolymer and hyaluronic acid (HA) formed a self-assembled structure, which provides a biocompatible 3D environment for the cells. Multicellular spheroids were formed on the optimized plate and were observed as cell migration from a spheroid within a 3D matrix, such as the invasive and metastatic cancer of TME. This study suggests a new 3D platform using polymer complexes and the importance of tumor complexities, including various cell types and microenvironments.

Peptide Blocking CTLA-4 and B7-1 Interaction.

Discovery of the B7 family immune checkpoints such as CTLA-4 (CD152), PD-1 (CD279), as well as their ligands B7-1 (CD80), B7-2 (CD86), B7-H1 (PD-L1, CD274), and B7-DC (PD-L2, CD273), has opened new possibilities for cancer immunotherapy using monoclonal antibodies (mAb). The blockade of inhibitory receptors (CTLA-4 and PD-1) with specific mAb results in the activation of cancer patients’ T lymphocytes and tumor rejection. However, the use of mAb in clinics has several limitations including side effects and cost of treatment. The development of new low-molecular compounds that block immune checkpoints’ functional activity can help to overcome some of these limitations. In this paper, we describe a synthetic peptide (p344) containing 14 amino acids that specifically interact with CTLA-4 protein. A 3D computer model suggests that this peptide binds to the 99MYPPPY104 loop of CTLA-4 protein and potentially blocks the contact of CTLA-4 receptor with B7-1 ligand. Experimental data confirm the peptide-specific interaction with CTLA-4 and its ability to partially block CTLA-4/B7-1 binding. The identified synthetic peptide can be used for the development of novel immune checkpoint inhibitors that can block CTLA-4 functional activity for cancer immunotherapy.

Investigation on the Interactions between Self-Assembled β-Sheet Peptide Nanofibers and Model Cell Membranes.

Self-assembled peptide nanofibers (NFs) obtained from β-sheet peptides conjugated with drugs, including antigenic peptides, have recently attracted significant attention. However, extensive studies on the interactions of β-sheet peptide NFs with model cell membranes have not been reported. In this study, we investigated the interactions between three types of NFs, composed of PEG-peptide conjugates with different ethylene glycol (EG) lengths (6-, 12- and 24-mer), and dipalmitoylphosphatidylcholine (DPPC) Langmuir membranes. When increasing the EG chain length, those interactions significantly decreased considering measurements in the presence of the NFs of: (i) changes in surface pressure of the DPPC Langmuir monolayers and (ii) surface pressure–area (π–A) compression isotherms of DPPC. Because the observed trend was similar to the EG length dependency with regard to cellular association and cytotoxicity of the NFs that was reported previously, the interaction of NFs with phospholipid membranes represented a crucial factor to determine the cellular association and toxicity of the NFs. In contrast to NFs, no changes were observed with varying EG chain length on the interaction of the building block peptide with the DPPC membrane. The results obtained herein can provide a design guideline on the formulation of β-sheet peptide NFs, which may broaden its potential.

A Broad-Spectrum Antiviral Peptide Blocks Infection of Viruses by Binding to Phosphatidylserine in the Viral Envelope.

The ongoing threat of viral infections and the emergence of antiviral drug resistance warrants a ceaseless search for new antiviral compounds. Broadly-inhibiting compounds that act on elements shared by many viruses are promising antiviral candidates. Here, we identify a peptide derived from the cowpox virus protein CPXV012 as a broad-spectrum antiviral peptide. We found that CPXV012 peptide hampers infection by a multitude of clinically and economically important enveloped viruses, including poxviruses, herpes simplex virus-1, hepatitis B virus, HIV-1, and Rift Valley fever virus. Infections with non-enveloped viruses such as Coxsackie B3 virus and adenovirus are not affected. The results furthermore suggest that viral particles are neutralized by direct interactions with CPXV012 peptide and that this cationic peptide may specifically bind to and disrupt membranes composed of the anionic phospholipid phosphatidylserine, an important component of many viral membranes. The combined results strongly suggest that CPXV012 peptide inhibits virus infections by direct interactions with phosphatidylserine in the viral envelope. These results reiterate the potential of cationic peptides as broadly-acting virus inhibitors.