Stimulus-Responsive Amino Acids Behind In Situ Assembled Bioactive Peptide Materials

In situ self-assembly of peptides into well-defined nanostructures represents one of versatile strategies for creation of bioactive materials within living cells with great potential in disease diagnosis and treatment. The intimate relationship between amino acid sequences and the assembling propensity of peptides has been thoroughly elucidated over the past few decades. This has inspired development of various controllable self-assembling peptide systems based on stimuli-responsive naturally occurring or non-canonical amino acids, including redox-, pH-, photo-, enzyme-responsive amino acids. This review attempts to summarize the recent progress achieved in manipulating in situ self-assembly of peptides by controllable reactions occurring to amino acids. We will highlight the systems containing non-canonical amino acids developed in our laboratory during the past few years, primarily including acid/enzyme-responsive 4-aminoproline, redox-responsive (seleno)methionine, and enzyme-responsive 2-nitroimidazolyl alanine. Utilization of the stimuli-responsive assembling systems in creation of bioactive materials will be specifically introduced to emphasize their advantages for addressing the concerns lying in disease theranostics. Eventually, we will provide the perspectives for the further development of stimulus-responsive amino acids and thereby demonstrating their great potential in development of next-generation biomaterials.     

In Silico Design of MDM2-Targeting Peptides from a Naturally Occurring Constrained Peptide

Naturally occurring constrained peptides are frequently used as scaffolds for bioactive peptide grating due to their high stability. Here, we used in silico methods to design several constrained peptides comprising a scorpion toxin scaffold, a MDM2 binding epitope, and a cluster of positively charged residues. The designed peptides displayed varied binding affinity to MDM2 despite differing by only one or two residues. One of the peptides, SC426, had nanomolar binding affinity (K_D=6.6±2.6 nm ) to MDM2, and exhibited stronger inhibitory activity on the proliferation of HCT116 cells (p53-wild type) and SW480 cells (p53-mutant) than that of nutlin-3a. Binding mode analysis of the designed peptide at MDM2 suggests that the conserved “FWL” epitope was buried in the hydrophobic binding pocket, and the residues located at the periphery of the binding site contributed to the high binding affinity of SC426. Overall, in silico design of miniproteins with therapeutic potential through epitope grafting to the naturally occurring constrained peptide is an effective strategy.     

Bioactive Peptides as Potential Nutraceuticals for Diabetes Therapy: A Comprehensive Review

Diabetes mellitus is a major public health concern associated with high mortality and reduced life expectancy. The alarming rise in the prevalence of diabetes is linked to several factors including sedentary lifestyle and unhealthy diet. Nutritional intervention and increased physical activity could significantly contribute to bringing this under control. Food-derived bioactive peptides and protein hydrolysates have been associated with a number health benefits. Several peptides with antidiabetic potential have been identified that could decrease blood glucose level, improve insulin uptake and inhibit key enzymes involved in the development and progression of diabetes. Dietary proteins, from a wide range of food, are rich sources of antidiabetic peptides. Thus, there are a number of benefits in studying peptides obtained from food sources to develop nutraceuticals. A deeper understanding of the underlying molecular mechanisms of these peptides will assist in the development of new peptide-based therapeutics. Despite this, a comprehensive analysis of the antidiabetic properties of bioactive peptides derived from various food sources is still lacking. Here, we review the recent literature on food-derived bioactive peptides possessing antidiabetic activity. The focus is on the effectiveness of these peptides as evidenced by in vitro and in vivo studies. Finally, we discuss future prospects of peptide-based drugs for the treatment of diabetes.     

Ribbon-like Foldamers for Cellular Uptake and Drug Delivery

Different intracellular delivery systems of bioactive compounds have been developed, including cell-penetrating peptides. Although usually nontoxic and biocompatible, these vectors share some of the general drawbacks of peptides, notably low bioavailability and susceptibility to protease degradation, that limit their use. Herein, the conversion of short peptide sequences into poly-α-amino-γ-lactam foldamers that adopt a ribbon-like structure is investigated. This template is used to distribute critical cationic and/or hydrophobic groups on both sides of the backbone, leading to potent short, cell-permeable foldamers with a low positive-charge content. The lead compound showed dramatically improved protease resistance and was able to efficiently deliver a biologically relevant cargo inside cells. This study provided a simple strategy to convert short peptide sequences into efficient protease-resistant cell-penetrating foldamers.     

Antioxidant and ACE Inhibitory Bioactive Peptides Purified from Egg Yolk Proteins

Protein by-products from the extraction of lecithin from egg yolk can be converted into value-added products, such as bioactive hydrolysates and peptides that have potential health enhancing antioxidant, and antihypertensive properties. In this study, the antioxidant and angiotensin converting enzyme (ACE) inhibitory activities of peptides isolated and purified from egg yolk protein were investigated. Defatted egg yolk was hydrolyzed using pepsin and pancreatin and sequentially fractionated by ultrafiltration, followed by gel filtration to produce egg yolk gel filtration fractions (EYGF). Of these, two fractions, EYGF-23 and EYGF-33, effectively inhibited the peroxides and thiobarbituric acid reactive substance (TBARS) in an oxidizing linoleic acid model system. The antioxidant mechanism involved superoxide anion and hydroxyl radicals scavenging and ferrous chelation. The presence of hydrophobic amino acids such as tyrosine (Y) and tryptophan (W), in sequences identified by LC-MS as WYGPD (EYGF-23) and KLSDW (EYGF-33), contributed to the antioxidant activity and were not significantly different from the synthetic BHA antioxidant. A third fraction (EYGF-56) was also purified from egg yolk protein by gel filtration and exhibited high ACE inhibitory activity (69%) and IC₅₀ value (3.35 mg/mL). The SDNRNQGY peptide (10 mg/mL) had ACE inhibitory activity, which was not significantly different from that of the positive control captopril (0.5 mg/mL). In addition, YPSPV in (EYGF-33) (10 mg/mL) had higher ACE inhibitory activity compared with captopril. These findings indicated a substantial potential for producing valuable peptides with antioxidant and ACE inhibitory activity from egg yolk.     

Role of Bioactive Peptide Sequences in the Potential Impact of Dairy Protein Intake on Metabolic Health

For years, there has been an increasing move towards elucidating the complexities of how food can interplay with the signalling networks underlying energy homeostasis and glycaemic control. Dairy foods can be regarded as the greatest source of proteins and peptides with various health benefits and are a well-recognized source of bioactive compounds. A number of dairy protein-derived peptide sequences with the ability to modulate functions related to the control of food intake, body weight gain and glucose homeostasis have been isolated and characterized. Their being active in vivo may be questionable mainly due to expected low bioavailability after ingestion, and hence their real contribution to the metabolic impact of dairy protein intake needs to be discussed. Some reports suggest that the differential effects of dairy proteins—in particular whey proteins—on mechanisms underlying energy balance and glucose-homeostasis may be attributed to their unique amino acid composition and hence the release of free amino acid mixtures enriched in essential amino acids (i.e., branched-chain-amino acids) upon digestion. Actually, the research reports reviewed in this article suggest that, among a number of dairy protein-derived peptides isolated and characterized as bioactive compounds in vitro, some peptides can be active in vivo post-oral administration through a local action in the gut, or, alternatively, a systemic action on specific molecular targets after entering the systemic circulation. Moreover, these studies highlight the importance of the enteroendocrine system in the cross talk between food proteins and the neuroendocrine network regulating energy balance.     

In Vivo and In Vitro Comparison of the DPP-IV Inhibitory Potential of Food Proteins from Different Origins after Gastrointestinal Digestion

Dipeptidyl-peptidase IV (DPP-IV) plays an essential role in glucose metabolism by inactivating incretins. In this context, food-protein-derived DPP-IV inhibitors are promising glycemic regulators which may act by preventing the onset of type 2 diabetes in personalized nutrition. In this study, the DPP-IV-inhibitory potential of seven proteins from diverse origins was compared for the first time in vitro and in vivo in rat plasma after the intestinal barrier (IB) passage of the indigested proteins. The DPP-IV-inhibitory potentials of bovine hemoglobin, caseins, chicken ovalbumin, fish gelatin, and pea proteins were determined in rat plasma thirty minutes after oral administration. In parallel, these proteins, together with bovine whey and gluten proteins, were digested using the harmonized INFOGEST protocol adapted for proteins. The DPP-IV half-maximal inhibitory concentration (IC₅₀) was determined in situ using Caco-2 cells. The DPP-IV-inhibitory activity was also measured after IB passage using a Caco2/HT29-MTX mixed-cell model. The peptide profiles were analyzed using reversed-phase high-performance liquid chromatography tandem mass spectrometry (RP-HPLC-MS/MS) with MS data bioinformatics management, and the IC₅₀ of the identified peptides was predicted in silico. The in vitro and in vivo DPP-IV-inhibitory activity of the proteins differed according to their origin. Vegetable proteins and hemoglobin yielded the highest DPP-IV-inhibitory activity in vivo. However, no correlation was found between the in vivo and in vitro results. This may be partially explained by the differences between the peptidome analysis and the in silico predictions, as well as the study complexity.     

DPP-IV Inhibitory Peptides GPF,IGL, and GGGW Obtained from Chicken Blood Hydrolysates

Blood is a meat by-product rich in proteins with properties that can be improved after hydrolysis, making it a sustainable alternative for use in the generation of bioactive peptides. The objective of this study was to identify dipeptidyl peptidase IV (DPP-IV) inhibitory peptides obtained from different chicken blood hydrolysates prepared using combinations of four different enzymes. Best results were observed for AP (2% Alcalase + 5% Protana Prime) and APP (2% Alcalase + 5% Protana Prime + 3% Protana UBoost) hydrolysates obtaining inhibition values of 60.55 and 53.61%, respectively, assayed at a concentration of 10 mg/mL. Free amino acids were determined to establish the impact of exopeptidase activity in the samples. A total of 79 and 12 sequences of peptides were identified by liquid chromatography and mass spectrometry in tandem (LC-MS/MS) in AP and APP samples, respectively. Nine of the identified peptides were established as potential DPP-IV inhibitory using in silico approaches and later synthesized for confirmation. Thus, peptides GPF, IGL, and GGGW showed good DPP-IV inhibitory activity with IC₅₀ values of 0.94, 2.22, and 2.73 mM, respectively. This study confirmed the potential of peptides obtained from chicken blood hydrolysates to be used as DPP-IV inhibitors and, therefore, in the control or modulation of type 2 diabetes.     

Proteolytic Cleavage of Bioactive Peptides and Protease-Activated Receptors in Acute and Post-Colitis

The protease activity in inflammatory bowel disease (IBD) and irritable bowel syndrome has been studied extensively using synthetic fluorogenic substrates targeting specific sets of proteases. We explored activities in colonic tissue from a 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colitis rat model by investigating the cleavage of bioactive peptides. Pure trypsin- and elastase-like proteases on the one hand and colonic tissue from rats with TNBS-induced colitis in the acute or post-inflammatory phase on the other, were incubated with relevant peptides to identify their cleavage pattern by mass spectrometry. An increased cleavage of several peptides was observed in the colon from acute colitis rats. The tethered ligand (TL) sequences of peptides mimicking the N-terminus of protease-activated receptors (PAR) 1 and 4 were significantly unmasked by acute colitis samples and these cleavages were positively correlated with thrombin activity. Increased cleavage of β-endorphin and disarming of the TL-sequence of the PAR3-based peptide were observed in acute colitis and linked to chymotrypsin-like activity. Increased processing of the enkephalins points to the involvement of proteases with specificities different from trypsin- or chymotrypsin-like enzymes. In conclusion, our results suggest thrombin, chymotrypsin-like proteases and a set of proteases with different specificities as potential therapeutic targets in IBD.     

In vitro cytotoxicity of monticellite based bioactive ceramic powder synthesized from boron derivative waste

The cytotoxicity of monticellite based bioactive ceramic powder, which was synthesized from boron derivative waste has been determined by in vitro assays of MTT, NRU, and JC-1 staining. The toxicity of powder on different mammalian cell lines (3T3-L1, HUVEC, CRL-2120) was evaluated at the concentrations of 10, 100, 200, 400 and 800?µg/mL to justify its potential for biomedical applications. The obtained results showed that monticellite based bioactive ceramic powder possesses not only bioactive feature but also biocompatible characteristic at the concentration range of 10–200?µg/mL. Hence, monticellite based bioactive ceramics have high potential as a bone graft substitute for bone void filling and coating applications.     

Cyclization of the Antimicrobial Peptide Gomesin with Native Chemical Ligation: Influences on Stability and Bioactivity

Gomesin is an 18‐residue peptide originally isolated from the hemocytes of the Brazilian spider Acanthoscurria gomesiana. A broad spectrum of bioactivities have been attributed to gomesin, including in vivo and in vitro cytotoxicity against tumour cells, antimicrobial, antifungal, anti‐Leishmania and antimalarial effects. Given the potential therapeutic applications of gomesin, it was of interest to determine if an engineered version with a cyclic backbone has improved stability and bioactivity. Cyclization has been shown to confer enhanced stability and activity to a range of bioactive peptides and, in the case of a cone snail venom peptide, confer oral activity in a pain model. The current study demonstrates that cyclization improves the in vitro stability of gomesin over a 24 hour time period and enhances cytotoxicity against a cancer cell line without being toxic to a noncancerous cell line. In addition, antimalarial activity is enhanced upon cyclization. These findings provide additional insight into the influences of backbone cyclization on the therapeutic potential of peptides.     

Functionalized Peptide Fibrils as a Scaffold for Active Substances in Wound Healing

Technological developments in the field of biologically active peptide applications in medicine have increased the need for new methods for peptide delivery. The disadvantage of peptides as drugs is their low biological stability. Recently, great attention has been paid to self-assembling peptides that can form fibrils. Such a formulation makes bioactive peptides more resistant to enzymatic degradation and druggable. Peptide fibrils can be carriers for peptides with interesting biological activities. These features open up prospects for using the peptide fibrils as long-acting drugs and are a valid alternative to conventional peptidic therapies. In our study, we designed new peptide scaffolds that are a hybrid of three interconnected amino acid sequences and are: pro-regenerative, cleavable by neutrophilic elastase, and fibril-forming. We intended to obtain peptides that are stable in the wound environment and that, when applied, would release a biologically active sequence. Our studies showed that the designed hybrid peptides show a high tendency toward regular fibril formation and are able to release the pro-regenerative sequence. Cytotoxicity studies showed that all the designed peptides were safe, did not cause cytotoxic effects and revealed a pro-regenerative potential in human fibroblast and keratinocyte cell lines. In vivo experiments in a dorsal skin injury model in mice indicated that two tested peptides moderately promote tissue repair in their free form. Our research proves that peptide fibrils can be a druggable form and a scaffold for active peptides.     

Prediction of Drug Synergism between Peptides and Antineoplastic Drugs Paclitaxel, 5-Fluorouracil,and Doxorubicin Using In Silico Approaches

Chemotherapy is the main treatment for most early-stage cancers; nevertheless, its efficacy is usually limited by drug resistance, toxicity, and tumor heterogeneity. Cell-penetrating peptides (CPPs) are small peptide sequences that can be used to increase the delivery rate of chemotherapeutic drugs to the tumor site, therefore contributing to overcoming these problems and enhancing the efficacy of chemotherapy. The drug combination is another promising strategy to overcome the aforementioned problems since the combined drugs can synergize through interconnected biological processes and target different pathways simultaneously. Here, we hypothesized that different peptides (P1–P4) could be used to enhance the delivery of chemotherapeutic agents into three different cancer cells (HT-29, MCF-7, and PC-3). In silico studies were performed to simulate the pharmacokinetic (PK) parameters of each peptide and antineoplastic agent to help predict synergistic interactions in vitro. These simulations predicted peptides P2–P4 to have higher bioavailability and lower T_max, as well as the chemotherapeutic agent 5-fluorouracil (5-FU) to have enhanced permeability properties over other antineoplastic agents, with P3 having prominent accumulation in the colon. In vitro studies were then performed to evaluate the combination of each peptide with the chemotherapeutic agents as well as to assess the nature of drug interactions through the quantification of the Combination Index (CI). Our findings in MCF-7 and PC-3 cancer cells demonstrated that the combination of these peptides with paclitaxel (PTX) and doxorubicin (DOXO), respectively, is not advantageous over a single treatment with the chemotherapeutic agent. In the case of HT-29 colorectal cancer cells, the combination of P2–P4 with 5-FU resulted in synergistic cytotoxic effects, as predicted by the in silico simulations. Taken together, these findings demonstrate that these CPP6-conjugates can be used as adjuvant agents to increase the delivery of 5-FU into HT-29 colorectal cancer cells. Moreover, these results support the use of in silico approaches for the prediction of the interaction between drugs in combination therapy for cancer.     

Peptides from the Intestinal Tract of Breast Milk-Fed Infants Have Antimicrobial and Bifidogenic Activity

For bioactive milk peptides to be relevant to infant health, they must be released by gastrointestinal proteolysis and resist further proteolysis until they reach their site of activity. The intestinal tract is the likeliest site for most bioactivities, but it is currently unknown whether bioactive milk peptides are present therein. The purpose of the present study was to identify antimicrobial and bifidogenic peptides in the infant intestinal tract. Milk peptides were extracted from infant intestinal samples, and the activities of the bulk peptide extracts were determined by measuring growth of Escherichia coli, Staphylococcus aureus, and Bifidobacterium longum spp. infantis after incubation with serial dilutions. The peptide profiles of active and inactive samples were determined by peptidomics analysis and compared to identify candidate peptides for bioactivity testing. We extracted peptides from 29 intestinal samples collected from 16 infants. Five samples had antimicrobial activity against S. aureus and six samples had bifidogenic activity for B. infantis. We narrowed down a list of 6645 milk peptides to 11 candidate peptides for synthesis, of which 6 fully inhibited E. coli and S. aureus growth at concentrations of 2500 and 3000 µg/mL. This study provides evidence for the potential bioactivity of milk peptides in the infant intestinal tract.     

Membrane Vesicles for Nanoencapsulated Sulforaphane Increased Their Anti-Inflammatory Role on an In Vitro Human Macrophage Model

At present, there is a growing interest in finding new non-toxic anti-inflammatory drugs to treat inflammation, which is a key pathology in the development of several diseases with considerable mortality. Sulforaphane (SFN), a bioactive compound derived from Brassica plants, was shown to be promising due to its anti-inflammatory properties and great potential, though its actual clinical use is limited due to its poor stability and bioavailability. In this sense, the use of nanocarriers could solve stability-related problems. In the current study, sulforaphane loaded into membrane vesicles derived from broccoli plants was studied to determine the anti-inflammatory potential in a human-macrophage-like in vitro cell model under both normal and inflammatory conditions. On the one hand, the release of SFN from membrane vesicles was modeled in vitro, and two release phases were stabilized, one faster and the other slower due to the interaction between SFN and membrane proteins, such as aquaporins. Furthermore, the anti-inflammatory action of sulforaphane-loaded membrane vesicles was demonstrated, as a decrease in interleukins crucial for the development of inflammation, such as TNF-α, IL-1β and IL-6, was observed. Furthermore, these results also showed that membrane vesicles by themselves had anti-inflammatory properties, opening the possibility of new lines of research to study these vesicles, not only as carriers but also as active compounds.     

Glycosylated Cell-Penetrating Peptides (GCPPs)

The cell membrane regulates the exchange of molecules and information with the external environment. However, this control barrier hinders the delivery of exogenous bioactive molecules that can be applied to correct cellular malfunctions. Therefore, the traffic of macromolecules across the cell membrane represents a great challenge for the development of the next generation of therapies and diagnostic methods. Cell-penetrating peptides are short peptide sequences capable of delivering a broad range of biomacromolecules across the cellular membrane. However, penetrating peptides still suffer from limitations, mainly related to their lack of specificity and potential toxicity. Glycosylation has emerged as a potential promising strategy for the biological improvement of synthetic materials. In this work we have developed a new convergent strategy for the synthesis of penetrating peptides functionalized with glycan residues by an oxime bond connection. The uptake efficiency and intracellular distribution of these glycopeptides have been systematically characterized by means of flow cytometry and confocal microscopy and in zebrafish animal models. The incorporation of these glycan residues into the peptide structure influenced the internalization efficiency and cellular toxicity of the resulting glycopeptide hybrids in the different cell lines tested. The results reported herein highlight the potential of the glycosylation of penetrating peptides to modulate their activity.     

In Vitro Antioxidant Properties of Berry Leaves and Their Inhibitory Effect on Lipid Peroxidation of Thigh Meat from Broiler Chickens

The present study is designed to evaluate the antioxidant compounds from bilberry, cranberry, and raspberry leaves in connection with their radical scavenging activities, and their potential in inhibiting the lipid peroxidation of thigh meat from broiler chickens. For this purpose, plant extracts are analyzed regarding the content in bioactive compounds, antioxidant properties analysis including 1,1‐diphenyl‐2‐picrylhydrazyl, superoxide, hydrogen peroxide, and hydroxyl radical‐scavenging activities. Lipid peroxidation is induced by an Fe⁺³/ascorbic acid system and the inhibitory effects of plants extracts are assessed using thiobarbituric reactive substances (TBARS) as biomarkers for quantifying lipid oxidation in meat. The overview of the analyzed antioxidant compounds shows that cranberry leaves present the highest concentrations of vitamin E, β‐carotene, lutein and zeaxanthin, and polyphenols and a total antioxidant capacity with 52% higher than bilberry and 360% than raspberry. Bilberry leaves extract shows the strongest effect (p  < 0.0001) in inhibiting meat lipid peroxidation. Practical Applications: The results demonstrate the positive effect of the selected berry leaves in the oxidative stability of broiler chicken meat under in vitro conditions, and may be suggested as an alternative source of natural antioxidants for animal nutrition, with benefits in inhibiting and delaying oxidation, and also enhancing the nutritional value of meat.     

In vivo evaluation of cerium,gallium and vanadium-doped borate-based bioactive glass scaffolds using rat subcutaneous implantation model

The main objective of this study was to evaluate the cerium, gallium and vanadium-containing bioactive borate glass scaffolds for soft tissue applications and determine the potential toxicity of these scaffolds on the adjacent tissues. The effects of the cerium, gallium and vanadium substitution on the soft tissue ingrowth and angiogenesis in porous borate based bioactive glass scaffolds were investigated using rat subcutaneous implantation model. For this purpose, bioactive borate glass powders containing therapeutic ions were prepared by melt-cast method and subsequently scaffolds were fabricated using polymer foam replication technique. The scaffolds were implanted subcutaneously for 4 weeks in Sprague Dawley rats. Bare borate glass scaffolds with the same microstructure were used as the control. Histology was used to evaluate tissue ingrowth and blood vessel formation in the implants. Additionally, the antibacterial activities of cerium, gallium and vanadium containing porous bioactive glass scaffolds were investigated in vitro by a zone inhibition method. Results revealed that addition of cerium ions to the borate glass network caused an increase in blood vessel formation. On the other hand, a decrease was obtained in angiogenesis in gallium and vanadium-containing glasses. All of the scaffolds prepared in the study did not show any antibacterial activity towards Escherichia coli and Staphylococcus aureus.     

Phosphorylation as a tool to modulate aggregation propensity and to predict fibril architecture

Despite the importance of post-translational modifications in controlling the solubility and conformational properties of proteins and peptides, precisely how the aggregation propensity of different peptide sequences is modulated by chemical modification remains unclear. Here we have investigated the effect of phosphorylation on the aggregation propensity of a 13-residue synthetic peptide incorporating one or more phosphate groups at seven different sites at various pH values. Fibril formation was shown to be inhibited when a single phosphate group was introduced at all seven locations in the peptide sequence at pH 7.5, when the phosphate group is fully charged. By contrast, when the same peptides were analysed at pH 1.1, when the phosphate is fully protonated, fibrils from all seven peptide sequences form rapidly. At intermediate pH values (pH 3.6) when the phosphate group is mono-anionic, the aggregation propensity of the peptides was found to be highly dependent on the position of the phosphate group in the peptide sequence. Using this information, combined with molecular dynamics (MD) simulations of the peptide sequence, we provide evidence consistent with the peptide forming amyloid fibrils with a class 7 architecture. The results highlight the potential utility of phosphorylation as a method of reversibly controlling the aggregation kinetics of peptide sequences both during and after synthesis. Moreover, by exploiting the ability of the phosphate group to adopt different charge states as a function of pH, and combining experimental insights with atomistic information calculated from MD simulations as pH is varied, we show how the resulting information can be used to predict fibril structures consistent with both datasets, and use these to rationalise their sensitivity of fibrillation kinetics both to the location of the phosphate group and its charge state.