The Peptide Functionalized Inorganic Nanoparticles for Cancer-Related Bioanalytical and Biomedical Applications

In order to improve their bioapplications, inorganic nanoparticles (NPs) are usually functionalized with specific biomolecules. Peptides with short amino acid sequences have attracted great attention in the NP functionalization since they are easy to be synthesized on a large scale by the automatic synthesizer and can integrate various functionalities including specific biorecognition and therapeutic function into one sequence. Conjugation of peptides with NPs can generate novel theranostic/drug delivery nanosystems with active tumor targeting ability and efficient nanosensing platforms for sensitive detection of various analytes, such as heavy metallic ions and biomarkers. Massive studies demonstrate that applications of the peptide–NP bioconjugates can help to achieve the precise diagnosis and therapy of diseases. In particular, the peptide–NP bioconjugates show tremendous potential for development of effective anti-tumor nanomedicines. This review provides an overview of the effects of properties of peptide functionalized NPs on precise diagnostics and therapy of cancers through summarizing the recent publications on the applications of peptide–NP bioconjugates for biomarkers (antigens and enzymes) and carcinogens (e.g., heavy metallic ions) detection, drug delivery, and imaging-guided therapy. The current challenges and future prospects of the subject are also discussed.     

A Global Review on Short Peptides: Frontiers and Perspectives

Peptides are fragments of proteins that carry out biological functions. They act as signaling entities via all domains of life and interfere with protein-protein interactions, which are indispensable in bio-processes. Short peptides include fundamental molecular information for a prelude to the symphony of life. They have aroused considerable interest due to their unique features and great promise in innovative bio-therapies. This work focusing on the current state-of-the-art short peptide-based therapeutical developments is the first global review written by researchers from all continents, as a celebration of 100 years of peptide therapeutics since the commencement of insulin therapy in the 1920s. Peptide “drugs” initially played only the role of hormone analogs to balance disorders. Nowadays, they achieve numerous biomedical tasks, can cross membranes, or reach intracellular targets. The role of peptides in bio-processes can hardly be mimicked by other chemical substances. The article is divided into independent sections, which are related to either the progress in short peptide-based theranostics or the problems posing challenge to bio-medicine. In particular, the SWOT analysis of short peptides, their relevance in therapies of diverse diseases, improvements in (bio)synthesis platforms, advanced nano-supramolecular technologies, aptamers, altered peptide ligands and in silico methodologies to overcome peptide limitations, modern smart bio-functional materials, vaccines, and drug/gene-targeted delivery systems are discussed.     

Peptide Targeting of PDZ-Dependent Interactions as Pharmacological Intervention in Immune-Related Diseases

PDZ (postsynaptic density (PSD95), discs large (Dlg), and zonula occludens (ZO-1)-dependent interactions are widely distributed within different cell types and regulate a variety of cellular processes. To date, some of these interactions have been identified as targets of small molecules or peptides, mainly related to central nervous system disorders and cancer. Recently, the knowledge of PDZ proteins and their interactions has been extended to various cell types of the immune system, suggesting that their targeting by viral pathogens may constitute an immune evasion mechanism that favors viral replication and dissemination. Thus, the pharmacological modulation of these interactions, either with small molecules or peptides, could help in the control of some immune-related diseases. Deeper structural and functional knowledge of this kind of protein–protein interactions, especially in immune cells, will uncover novel pharmacological targets for a diversity of clinical conditions.     

In Silico Analysis and In Vitro Characterization of the Bioactive Profile of Three Novel Peptides Identified from 19 kDa α-Zein Sequences of Maize

In this study, we characterized three novel peptides derived from the 19 kDa α-zein, and determined their bioactive profile in vitro and developed a structural model in silico. The peptides, 19ZP1, 19ZP2 and 19ZP3, formed α-helical structures and had positive and negative electrostatic potential surfaces (range of −1 to +1). According to the in silico algorithms, the peptides displayed low probabilities for cytotoxicity (≤0.05%), cell penetration (10–33%) and antioxidant activities (9–12.5%). Instead, they displayed a 40% probability for angiotensin-converting enzyme (ACE) inhibitory activity. For in vitro characterization, peptides were synthesized by solid phase synthesis and tested accordingly. We assumed α-helical structures for 19ZP1 and 19ZP2 under hydrophobic conditions. The peptides displayed antioxidant activity and ACE-inhibitory activity, with 19ZP1 being the most active. Our results highlight that the 19 kDa α-zein sequences could be explored as a source of bioactive peptides, and indicate that in silico approaches are useful to predict peptide bioactivities, but more structural analysis is necessary to obtain more accurate data.     

Molecular Dynamics Scoring of Protein–Peptide Models Derived from Coarse-Grained Docking

One of the major challenges in the computational prediction of protein–peptide complexes is the scoring of predicted models. Usually, it is very difficult to find the most accurate solutions out of the vast number of sometimes very different and potentially plausible predictions. In this work, we tested the protocol for Molecular Dynamics (MD)-based scoring of protein–peptide complex models obtained from coarse-grained (CG) docking simulations. In the first step of the scoring procedure, all models generated by CABS-dock were reconstructed starting from their original C-alpha trace representations to all-atom (AA) structures. The second step included geometry optimization of the reconstructed complexes followed by model scoring based on receptor–ligand interaction energy estimated from short MD simulations in explicit water. We used two well-known AA MD force fields, CHARMM and AMBER, and a CG MARTINI force field. Scoring results for 66 different protein–peptide complexes show that the proposed MD-based scoring approach can be used to identify protein–peptide models of high accuracy. The results also indicate that the scoring accuracy may be significantly affected by the quality of the reconstructed protein receptor structures.     

Uptake of Cell-Penetrating Peptide RL2 by Human Lung Cancer Cells: Monitoring by Electron Paramagnetic Resonance and Confocal Laser Scanning Microscopy

RL2 is a recombinant analogue of a human κ-casein fragment, capable of penetrating cells and inducing apoptosis of cancer cells with no toxicity to normal cells. The exact mechanism of RL2 penetration into cells remains unknown. In this study, we investigated the mechanism of RL2 penetration into human lung cancer A549 cells by a combination of electron paramagnetic resonance (EPR) spectroscopy and confocal laser scanning microscopy. EPR spectra of A549 cells incubated with RL2 (sRL2) spin-labeled by a highly stable 3-carboxy-2,2,5,5-tetraethylpyrrolidine-1-oxyl radical were found to contain three components, with their contributions changing with time. The combined EPR and confocal-microscopy data allowed us to assign these three forms of sRL2 to the spin-labeled protein sticking to the membrane of the cell and endosomes, to the spin-labeled protein in the cell interior, and to spin labeled short peptides formed in the cell because of protein digestion. EPR spectroscopy enabled us to follow the kinetics of transformations between different forms of the spin-labeled protein at a minimal spin concentration (3–16 μM) in the cell. The prospects of applications of spin-labeled cell-penetrating peptides to EPR imaging, DNP, and magnetic resonance imaging are discussed, as is possible research on an intrinsically disordered protein in the cell by pulsed dipolar EPR spectroscopy.     

Near-Wall Aggregation of Amyloidogenic Aβ 1-40 Peptide: Direct Observation by the FRET

The formation of amyloid fibrils is one of the variants of the self-organization of polypeptide chains. For the amyloid aggregation, the solution must be oversaturated with proteins. The interface of the liquid (solution) and solid (vessel walls) phases can trigger the adsorption of protein molecules, and the resulting oversaturation can initiate conformational transitions in them. In any laboratory experiment, we cannot exclude the presence of surfaces such as the walls of vessels, cuvettes, etc. However, in many works devoted to the study of amyloid formation, this feature is not considered. In our work, we investigated the behavior of the Aβ 1-40 peptide at the water–glass, water–quartz, and water–plastic interface. We carried out a series of simple experiments and showed that the Aβ 1-40 peptide is actively adsorbed on these surfaces, which leads to a significant interaction and aggregation of peptides. This means that the interface can be the place where the first amyloid nucleus appears. We suggest that this effect may also be one of the reasons for the difficulty of reproducing kinetic data when studying the aggregation of the amyloid of the Aβ 1-40 peptide and other amyloidogenic proteins     

Diphenylalanine Motif Drives Self-Assembling in Hybrid PNA-Peptide Conjugates

Peptides and nucleic acids can self-assemble to give supramolecular structures that find application in different fields, ranging from the delivery of drugs to the obtainment of materials endowed with optical properties. Forces that stabilize the “suprastructures” typically are hydrogen bonds or aromatic interactions; in case of nucleic acids, Watson-Crick pairing drives self-assembly while, in case of peptides, backbone hydrogen bonds and interactions between aromatic side chains trigger the formation of structures, such as nanotubes or ribbons. Molecules containing both aromatic peptides and nucleic acids could in principle exploit different forces to self-assemble. In this work we meant to investigate the self-assembly of mixed systems, with the aim to understand which forces play a major role and determine formation/structure of aggregates. We therefore synthesized conjugates of the peptide FF to the peptide nucleic acid dimer “gc” and characterized their aggregates by different spectroscopic techniques, including NMR, CD and fluorescence.     

用于基因和药物传递的多肽及聚多肽材料

多肽和聚多肽作为一类新型的生物医用材料,由于其具有良好的生物活性、生物可降解性以及生物相容性而备受瞩目.将具有特殊生理功能的多肽作为基因或药物载体、或用于药物修饰等,可以提高基因转染效率,增强药物的靶向治疗效果,降低药物的毒副作用.本文综述了近年来多肽及聚多肽材料在这些生物医学领域的应用及进展,对部分活性肽的作用机制和...     

Identification,Characterization and Antihypertensive Effect In Vivo of a Novel ACE-Inhibitory Heptapeptide from Defatted Areca Nut Kernel Globulin Hydrolysates

The areca (Areca catechu L.) nut kernel (ANK) is a good potential protein source for its high protein content of 9.89–14.62 g/100 g and a high yield of around 300,000 tons per year in China. However, utilization of the areca nut kernel is limited. To expand the usage of ANK in pharmaceutical or foods industries, areca nut kernel globulin was extracted and angiotensin-I converting enzyme (ACE) inhibition peptides were prepared and identified using gel chromatography, reversed phase HPLC separation, UPLC-ESI-MS/MS analysis and in silico screening. Finally, a novel ACE-inhibitory heptapeptide (Ala–Pro–Lys–Ile–Glu–Glu–Val) was identified and chemically synthesized. The combination pattern between APKIEEV and ACE, and the inhibition kinetics, antihypertensive effect and endothlein-1 inhibition activity of APKIEEV were studied. The results of the molecular docking demonstrated that APKIEEV could bind to four active sites (not the key active sites) of ACE via short hydrogen bonds and demonstrated high ACE-inhibitory activity (IC₅₀: 550.41 μmol/L). Moreover, APKIEEV exhibited a significantly lowering effect on both the systolic blood pressure and diastolic blood pressure of spontaneously hypertensive rats, and had considerable suppression ability on intracellular endothelin-1. These results highlight the potential usage of APKIEEV as ingredients of antihypertensive drugs or functional foods.     

Reactive Acrylamide-Modified DNA Traps for Accurate Cross-Linking with Cysteine Residues in DNA–Protein Complexes Using Mismatch Repair Protein MutS as a Model

Covalent protein capture (cross-linking) by reactive DNA derivatives makes it possible to investigate structural features by fixing complexes at different stages of DNA–protein recognition. The most common cross-linking methods are based on reactive groups that interact with native or engineered cysteine residues. Nonetheless, high reactivity of most of such groups leads to preferential fixation of early-stage complexes or even non-selective cross-linking. We synthesised a set of DNA reagents carrying an acrylamide group attached to the C5 atom of a 2′-deoxyuridine moiety via various linkers and studied cross-linking with MutS as a model protein. MutS scans DNA for mismatches and damaged nucleobases and can form multiple non-specific complexes with DNA that may cause non-selective cross-linking. By varying the length of the linker between DNA and the acrylamide group and by changing the distance between the reactive nucleotide and a mismatch in the duplex, we showed that cross-linking occurs only if the distance between the acrylamide group and cysteine is optimal within the DNA–protein complex. Thus, acrylamide-modified DNA duplexes are excellent tools for studying DNA–protein interactions because of high selectivity of cysteine trapping.     

Synthetic Channel-forming Peptides and Ion Selectivity

Introduction PeptidesmadeupofalternatingL andD amino acidscanformβhelicesasingramicidinAorcyclic peptidesthataggregatetoformtubes[1].Inbothcases thestructuresarehollowwithallthesidechainspro jectingoutwards.Kennedyetal.[2]postulatedthat peptideshavingthe…     

Design and Characterisation of Inhibitory Peptides against Bleg1_2478, an Evolutionary Divergent B3 Metallo-β-lactamase

Previously, a hypothetical protein (HP) termed Bleg1_2437 (currently named Bleg1_2478) from Bacillus lehensis G1 was discovered to be an evolutionary divergent B3 subclass metallo-β-lactamase (MBL). Due to the scarcity of clinical inhibitors for B3 MBLs and the divergent nature of Bleg1_2478, this study aimed to design and characterise peptides as inhibitors against Bleg1_2478. Through in silico docking, RSWPWH and SSWWDR peptides with comparable binding energy to ampicillin were obtained. In vitro assay results showed RSWPWH and SSWWDR inhibited the activity of Bleg1_2478 by 50% at concentrations as low as 0.90 µM and 0.50 µM, respectively. At 10 µM of RSWPWH and 20 µM of SSWWDR, the activity of Bleg1_2478 was almost completely inhibited. Isothermal titration calorimetry (ITC) analyses showed slightly improved binding properties of the peptides compared to ampicillin. Docked peptide–protein complexes revealed that RSWPWH bound near the vicinity of the Bleg1_2478 active site while SSWWDR bound at the center of the active site itself. We postulate that the peptides caused the inhibition of Bleg1_2478 by reducing or blocking the accessibility of its active site from ampicillin, thus hampering its catalytic function.     

Modulating Protein–Protein Interactions by Cyclic and Macrocyclic Peptides. Prominent Strategies and Examples

Cyclic and macrocyclic peptides constitute advanced molecules for modulating protein–protein interactions (PPIs). Although still peptide derivatives, they are metabolically more stable than linear counterparts, and should have a lower degree of flexibility, with more defined secondary structure conformations that can be adapted to imitate protein interfaces. In this review, we analyze recent progress on the main methods to access cyclic/macrocyclic peptide derivatives, with emphasis in a few selected examples designed to interfere within PPIs. These types of peptides can be from natural origin, or prepared by biochemical or synthetic methodologies, and their design could be aided by computational approaches. Some advances to facilitate the permeability of these quite big molecules by conjugation with cell penetrating peptides, and the incorporation of β-amino acid and peptoid structures to improve metabolic stability, are also commented. It is predicted that this field of research could have an important future mission, running in parallel to the discovery of new, relevant PPIs involved in pathological processes.     

Target-templated de novo design of macrocyclic d-/l-peptides: discovery of drug-like inhibitors of PD-1

Peptides are a rapidly growing class of therapeutics with various advantages over traditional small molecules, especially for targeting difficult protein–protein interactions. However, current structure-based methods are largely limited to natural peptides and are not suitable for designing bioactive cyclic topologies that go beyond natural l-amino acids. Here, we report a generalizable framework that exploits the computational power of Rosetta, in terms of large-scale backbone sampling, side-chain composition and energy scoring, to design heterochiral cyclic peptides that bind to a protein surface of interest. To showcase the applicability of our approach, we developed two new inhibitors (PD-i3 and PD-i6) of programmed cell death 1 (PD-1), a key immune checkpoint in oncology. A comprehensive biophysical evaluation was performed to assess their binding to PD-1 as well as their blocking effect on the endogenous PD-1/PD-L1 interaction. Finally, NMR elucidation of their in-solution structures confirmed our de novo design approach.

In silico design of heterochiral cyclic peptides that bind to a specific surface patch on the target protein (PD-1, in this case) and disrupt protein–protein interactions.     

Effects of acidic peptide size and sequence on trivalent praseodymium adduction and electron transfer dissociation mass spectrometry

Using the lanthanide ion praseodymium, Pr(III), metallated ion formation and electron transfer dissociation (ETD) were studied for 25 biological and model acidic peptides. For chain lengths of seven or more residues, even highly acidic peptides that can be difficult to protonate by electrospray ionization will metallate and undergo abundant ETD fragmentation. Peptides composed of predominantly acidic residues form only the deprotonated ion, [M + Pr ‐ H]²⁺; this ion yields near complete ETD sequence coverage for larger peptides. Peptides with a mixture of acidic and neutral residues generate [M + Pr]³⁺, which cleaves between every residue for many peptides. Acidic peptides that contain at least one residue with a basic side chain also produce the protonated ion, [M + Pr + H]⁴⁺; this ion undergoes the most extensive sequence coverage by ETD. Primarily metallated and non‐metallated c‐ and z‐ions form for all peptides investigated. Metal adducted product ions are only present when at least half of the peptide sequence can be incorporated into the ion; this suggests that the metal ion simultaneously attaches to more than one acidic site. The only site consistently lacking dissociation is at the N‐terminal side of a proline residue. Increasing peptide chain length generates more backbone cleavage for metal‐peptide complexes with the same charge state. For acidic peptides with the same length, increasing the precursor ion charge state from 2+ to 3+ also leads to more cleavage. The results of this study indicate that highly acidic peptides can be sequenced by ETD of complexes formed with Pr(III). Copyright © 2017 John Wiley & Sons, Ltd.     

A New Class of Uracil–DNA Glycosylase Inhibitors Active against Human and Vaccinia Virus Enzyme

Uracil–DNA glycosylases are enzymes that excise uracil bases appearing in DNA as a result of cytosine deamination or accidental dUMP incorporation from the dUTP pool. The activity of Family 1 uracil–DNA glycosylase (UNG) activity limits the efficiency of antimetabolite drugs and is essential for virulence in some bacterial and viral infections. Thus, UNG is regarded as a promising target for antitumor, antiviral, antibacterial, and antiprotozoal drugs. Most UNG inhibitors presently developed are based on the uracil base linked to various substituents, yet new pharmacophores are wanted to target a wide range of UNGs. We have conducted virtual screening of a 1,027,767-ligand library and biochemically screened the best hits for the inhibitory activity against human and vaccinia virus UNG enzymes. Although even the best inhibitors had IC₅₀ ≥ 100 μM, they were highly enriched in a common fragment, tetrahydro-2,4,6-trioxopyrimidinylidene (PyO3). In silico, PyO3 preferably docked into the enzyme’s active site, and in kinetic experiments, the inhibition was better consistent with the competitive mechanism. The toxicity of two best inhibitors for human cells was independent of the presence of methotrexate, which is consistent with the hypothesis that dUMP in genomic DNA is less toxic for the cell than strand breaks arising from the massive removal of uracil. We conclude that PyO3 may be a novel pharmacophore with the potential for development into UNG-targeting agents.     

Tandem trimer pyrrole–imidazole polyamide probes targeting 18 base pairs in human telomere sequences

The binding of molecules to specific DNA sequences is important for imaging genome DNA and for studying gene expression. Increasing the number of base pairs targeted by these molecules would provide greater specificity. N-Methylpyrrole–N-methylimidazole (Py–Im) polyamides are one type of such molecules and can bind to the minor groove of DNA in a sequence-specific manner without causing denaturation of DNA. Our recent work has demonstrated that tandem hairpin Py–Im polyamides conjugated with a fluorescent dye can be synthesized easily and can serve as new probes for studying human telomeres under mild conditions. Herein, to improve their selectivities to telomeres by targeting longer sequences, we designed and synthesized a fluorescent tandem trimer Py–Im polyamide probe, comprising three hairpins and two connecting regions (hinges). The new motif bound to 18 bp dsDNA in human telomeric repeats (TTAGGG)_n, the longest sequence for specific binding reported for Py–Im polyamides. We compared the binding affinities and the abilities to discriminate mismatch, the UV-visible absorption and fluorescence spectra, and telomere staining in human cells between the tandem trimer and a previously developed tandem hairpin. We found that the tandem trimer Py–Im polyamide probe has higher ability to recognize telomeric repeats and stains telomeres in chemically fixed cells with lower background signal.     

New Scabimycins A-C Isolated from Streptomyces acidiscabies (Lu19992)

Peptide natural products displaying a wide range of biological activities have become important drug candidates over the years. Microorganisms have been a powerful source of such bioactive peptides, and Streptomyces have yielded many novel natural products thus far. In an effort to uncover such new, meaningful compounds, the metabolome of Streptomyces acidiscabies was analyzed thoroughly. Three new compounds, scabimycins A–C (1–3), were discovered, and their chemical structures were elucidated by NMR spectroscopy. The relative and absolute configurations were determined using ROESY NMR experiments and advanced Marfey’s method.     

The Activating Effect of Strong Acid for Pd-Catalyzed Directed C–H Activation by Concerted Metalation-Deprotonation Mechanism

A computational study on the origin of the activating effect for Pd-catalyzed directed C–H activation by the concerted metalation-deprotonation (CMD) mechanism is conducted. DFT calculations indicate that strong acids can make Pd catalysts coordinate with directing groups (DGs) of the substrates more strongly and lower the C–H activation energy barrier. For the CMD mechanism, the electrophilicity of the Pd center and the basicity of the corresponding acid ligand for deprotonating the C–H bond are vital to the overall C–H activation energy barrier. Furthermore, this rule might disclose the role of some additives for C–H activation.