The established cancer treatment strategy in clinical setting is based on chemo and radiation therapy, having limitations due to severe side-effects and drug-resistance. Small molecule chemo-drugs target any fast-dividing cells irrespective of healthy or defective origin. As a result, a substantial amount of healthy tissue is also destroyed. Moreover, failure to recognize the heterogeneity of tumour tissue results in drug-resistance over the course of time. On the other hand, peptides and proteins actively target somatic changes that are signature to any specific tumour tissue. Development and metastasis of cancer cells require unique disruption/alteration of protein activity. Identification of those wild and cancerous genotypes and phenotypes is the key to establishing easy 'targets' for protein based targeted therapeutics. The approach is cytostatic and tissue specific, which reduces drug toxicity. Biopharmaceutical products based on proteins and peptides are slowly re-directing oncology from cytotoxic small molecular treatment approach to target oriented cytostatic strategy. This review focuses on current and upcoming peptide and protein-based precision therapeutics. At the same time, the study also shades light on the technological advancement in the field of protein and peptide-based therapeutics.
Therapeutic peptides derived proteins with alpha-reconformation states like antibody shape have shown potential effects in combating terrible diseases linked with earlier signs of angiogensis, mutagenesis and transgenesis. Alpha reconformation in material design refers to the folding of the peptide chains and their transitions under reversible chemical bonds of disulfide chemical bridges and further non-covalence lesions. Thus, the rational design of signal peptides into alpha-helix is intended in increasing the defending effects of peptides into cores like adjuvant antibiotic and/or vaccines. Thereby, the signal peptides are able in displaying multiple eradicating regions by changing crystal-depositions and deviation angles. These types of molecular structures could have multiple advantages in tracing disease syndromes and impurities by increasing the host defense against the fates of pathogens and viruses, eventually leading to the loss in signaling by increasing peptide susceptibility levels to folding and unfolding and therefore, formation of transgenic peptide models. Alpha reconformation peptides is aimed in triggering as well as other regulatory functions such as remodulating metabolic chain disorders of lipolysis and glucolysis by increasing the insulin and leptin resistance for best lipid storages and lipoprotein density distributions.
Bacterial and fungal infections are challenging due to their low susceptibility and resistance to antimicrobial drugs. For this reason, antimicrobial peptides (AMP) emerge as excellent alternatives to overcome these problems. At the same time, their active insertion into the cell wall of microorganisms can be availed for biorecognition applications in biosensing platforms. Temporin-PTA (T-PTA) is an AMP found in the skin secretions of the Malaysian fire frog Hylarana picturata, which presents antibacterial activity against MRSA, Escherichia coli, and Bacillus subtilis. In this work, T-PTA was explored as an innovative sensing layer aiming for the electrochemical differentiation of Klebsiella pneumoniae, Acinetobacter baumannii, Bacillus subtilis, Enterococcus faecalis, Candida albicans, and C. tropicalis based on the structural differences of their membranes. The biosensor was analyzed through electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). In this approach, the different structural features of each microorganism resulted in different adherence degrees and, therefore, different electrochemical responses. The transducing layer was fabricated by the self-assembling of a 4-mercaptobenzoic acid (MBA) monolayer and gold-capped magnetic nanoparticles (Fe3O4@Au) implemented to improve the electrical signal of the biointeraction. We found that each interaction, expressed in variations of electron transfer resistance and anodic peak current, demonstrated a singular response from which the platform can discriminate all different microorganisms. We found expressive sensitivity towards Gram-negative species, especially K. pneumoniae. A detection limit of 101 CFU.mL-1 and a linear range of 101 to 105 CFU.mL-1 were obtained. The T-PTA biosensor platform is a promising and effective tool for microbial identification.
Cancer is one of the leading causes of mortality worldwide; nearly 10 million people died from it in 2020. The high mortality rate results from the lack of effective screening approaches where early detection cannot be achieved, reducing the chance of early intervention to prevent cancer development. Non-invasive and deep-tissue imaging is useful in cancer diagnosis, contributing to a visual presentation of anatomy and physiology in a rapid and safe manner. Its sensitivity and specificity can be enhanced with the application of targeting ligands with the conjugation of imaging probes. Phage display is a powerful technology to identify antibody- or peptide-based ligands with effective binding specificity against their target receptor. Tumour-targeting peptides exhibit promising results in molecular imaging, but the application is limited to animals only. Modern nanotechnology facilitates the combination of peptides with various nanoparticles due to their superior characteristics, rendering novel strategies in designing more potent imaging probes for cancer diagnosis and targeting therapy. In the end, a myriad of peptide candidates that aimed for different cancers diagnosis and imaging in various forms of research were reviewed.
Targeting vascular endothelial growth factor receptor (VEFGR) and its co-receptor neuropilin-1 (NRP-1) is an interesting vascular strategy. tLyp-1 is a tumor-homing and penetrating peptide of 7 amino acids (CGNKRTR). It is a truncated form of Lyp-1 (CGNKRTRGC), which is known to target NRP-1 receptor, with high affinity and specificity. It is mediated by endocytosis via C-end rule (CendR) internalization pathway. The aim of this study is to evaluate the importance of each amino acid in the tLyp-1 sequence through alanine-scanning (Ala-scan) technique, during which each of the amino acid in the sequence was systematically replaced by alanine to produce 7 different analogues. In silico approach through molecular docking and molecular dynamics are employed to understand the interaction between the peptide and its analogues with the NRP-1 receptor, followed by in vitro ligand binding assay study. The C-terminal Arg is crucial in the interaction of tLyp-1 with NRP-1 receptor. Substituting this residue dramatically reduces the affinity of this peptide which is clearly seen in this study. Lys-4 is also important in the interaction, which is confirmed via the in vitro study and the MM-PBSA analysis. The finding in this study supports the CendR, in which the presence of R/K-XX-R/K motif is essential in the binding of a ligand with NRP-1 receptor. This presented work will serve as a guide in the future work pertaining the development of active targeting agent towards NRP-1 receptor.
The aim of this study was to produce and characterise nanosize liposomes containing bioactive peptides with antioxidative and ACE-inhibitory properties, derived from winged bean seeds (WBS) protein. WBS powder was papain-proteolysed, at 70 °C and pH 6.5 for six hours, followed by encapsulation via a solvent-free heating method. The results showed that the WBS proteolysate was successfully incorporated into spherical, unilamellar liposomal particles, with particle diameter, polydispersity index, zeta potential and encapsulation efficiency of 193.3 ± 0.12 nm, 0.4 ± 0.02 (unit less), -70.5 ± 0.30 mV and 27.6 ± 1.17%, respectively. It also demonstrated good storage stability over eight weeks at 4 °C, indicated by slight increment (15.1%) in particle size and a zeta potential only weaker by 17.2% at the end of the study period. These results suggested the feasibility of entrapping water soluble peptides in hydrophobic liposomal system that, upon optimisation, has the potential to act as bioactive food ingredient.
The food and health applications of bioactive peptides have grown remarkably in the past few decades. Current elucidations have shown that bioactive peptides have unique structural arrangement of amino acids, conferring distinct functionalities, and molecular affinity characteristics. However, whereas interest in the biological potency of bioactive peptides has grown, cost-effective techniques for monitoring the structural changes in these peptides and how these changes affect the biological properties have not grown at the same rate. Due to the high binding affinity of aptamers for other biomolecules, they have a huge potential for use in tracking the structural, conformational, and compositional changes in bioactive peptides. This review provides an overview of bioactive peptides and their essential structure-activity relationship. The review further highlights on the types and methods of synthesis of aptamers before the discussion of the prospects, merits, and challenges in the use of aptamers for bioaffinity interactions with bioactive peptides.
Application of non-thermal treatment to proteins prior to enzymatic hydrolysis can facilitate the release of novel bioactive peptides (BPs) with unique biological activities. In this study, lupin protein isolate was pre-treated with ultrasound and hydrolysed using alcalase and flavourzyme to produce alcalase hydrolysate (ACT) and flavourzyme hydrolysate(FCT). These hydrolysates were fractionated into 1, 5, and 10 kDa molecular weight fractions using a membrane ultrafiltration technique. The in vitro angiotensin-converting enzyme (ACE) studies revealed that unfractionated ACT (IC50 = 3.21 mg mL-1) and FCT (IC50 = 3.32 mg mL-1) were more active inhibitors of ACE in comparison to their ultrafiltrated fractions with IC50 values ranging from 6.09 to 7.45 mg mL-1. Molecular docking analysis predicted three unique peptides from ACT (AIPPGIPY, SVPGCT, and QGAGG) and FCT (AIPINNPGKL, SGNQGP, and PPGIP) as potential ACE inhibitors. Thus, unique BPs with ACE inhibitory effects might be generated from ultrasonicated lupin protein.
Dengue infections pose a critical threat to public health worldwide. Since there are no clinically approved antiviral drugs to treat dengue infections caused by the four dengue virus (DENV) serotypes, there is an urgent need to develop effective antivirals. Peptides are promising antiviral candidates due to their specificity and non-toxic properties. The DENV envelope (E) protein was selected for the design of antiviral peptides due to its importance in receptor binding and viral fusion to the host cell membrane. Twelve novel peptides were designed to mimic regions containing critical amino acid residues of the DENV E protein required for interaction with the host. A total of four peptides were identified to exhibit potent inhibitory effects against at least three or all four DENV serotypes. Peptide 3 demonstrated all three modes of action: cell protection and inhibition of post-infection against all four DENV serotypes, whereas direct virus-inactivating effects were only observed against DENV-2, 3, and 4. Peptide 4 showed good direct virus-inactivating effects against DENV-2 (74.26%) as well as good inhibitions of DENV-1 (80.37%) and DENV-4 (72.22%) during the post-infection stage. Peptide 5 exhibited direct virus-inactivating effects against all four DENV serotypes, albeit at lower inhibition levels against DENV-1 and DENV-3. It also exhibited highly significant inhibition of DENV-4 (89.31%) during post-infection. Truncated peptide 5F which was derived from peptide 5 showed more significant inhibition of DENV-4 (91.58%) during post-infection and good direct virus-inactivating effects against DENV-2 (77.55%) at a lower concentration of 100 μM. Peptide 3 could be considered as the best antiviral candidate for pre- and post-infection treatments of DENV infections in regions with four circulating dengue serotypes. However, if the most predominant dengue serotype for a particular region could be identified, peptides with significantly high antiviral activities against that particular dengue serotype could serve as more suitable antiviral candidates. Thus, peptide 5F serves as a more suitable antiviral candidate for post-infection treatment against DENV-4.
Dengue presents a major public health concern in over 100 countries due to the absence of an effective vaccine and antiviral therapy against all four dengue virus (DENV) serotypes. Several antiviral peptides were previously reported to inhibit at least three or all four DENV serotypes. Chemical modifications such as d-amino acid substitutions, polyethylene glycol (PEG)ylation, and cyclization could be applied to peptides to improve their biological activities and stability in serum. The PEGylated peptide 3 (PEG-P3) was identified to be the most promising antiviral candidate as it demonstrated good inhibitory effects against all four DENV serotypes during the pre- and post-infection stages, Based on the RP-HPLC and LC/MS analysis, peptide 4 was identified to be more stable in human serum than peptide 3, with 78.9 % and 41.6 % of the peptides remaining after 72 h of incubation in human serum, respectively. Both peptides were also able to retain their antiviral activities against specific DENV serotypes after 72 h incubation in human serum. PEG-P3 was found to be more stable than the unmodified peptide 3 with 89.4 % of PEG-P3 remaining in the human serum after 72 h of incubation. PEG-P3 was able to retain its inhibitory effects against DENV-1 to 4 after 72 h of incubation in human serum. This study provided insights into the antiviral activities and stabilities of the unmodified and chemically modified peptides in human serum.
The study was aimed to differentiate between porcine and bovine gelatines in adulterated samples by utilising sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) combined with principal component analysis (PCA). The distinct polypeptide patterns of 6 porcine type A and 6 bovine type B gelatines at molecular weight ranged from 50 to 220 kDa were studied. Experimental samples of raw gelatine were prepared by adding porcine gelatine in a proportion ranging from 5% to 50% (v/v) to bovine gelatine and vice versa. The method used was able to detect 5% porcine gelatine added to the bovine gelatine. There were no differences in the electrophoretic profiles of the jelly samples when the proteins were extracted with an acetone precipitation method. The simple approach employing SDS-PAGE and PCA reported in this paper may provide a useful tool for food authenticity issues concerning gelatine.
The application of proteomic and peptidomic technologies for food-derived bioactive peptides is an emerging field in food sciences. These technologies include the use of separation tools coupled to a high-resolution spectrometric and bioinformatic tools for prediction, identification, sequencing, and characterization of peptides. To a large extent, one-dimensional separation technologies have been extensively used as a continuous tool under different optimized conditions for the identification and analysis of food peptides. However, most one-dimensional separation technologies are fraught with significant bottlenecks such as insufficient sensitivity and specificity limits for complex samples. To address this limitation, separation systems based on orthogonal, multidimensional principles, which allow for the coupling of more than one analytical separation tool with different operational principles, provide a higher separation power than one-dimensional separation tools. This review describes the structure-informed separation and purification of protein hydrolyzates to obtain peptides with desirable bioactivities. PRACTICAL APPLICATIONS: Application of bioactive peptides in the formulation of functional foods, nutraceuticals, and therapeutic agents have increasingly gained scholarly and industrial attention. The bioactive peptides exist originally in protein sources and are only active after hydrolysis of the parent protein. Currently, several tools can be configured in one-dimensional or multidimensional systems for the separation and purification of protein hydrolyzates. The separations are informed by the structural properties such as the molecular weight, charge, hydrophobicity or hydrophilicity, and the solubility of peptides. This review provides a concise discussion on the commonly used analytical tools, their configurations, advantages and challenges in peptide separation. Emphasis is placed on how the structural properties of peptides assist in the separation and purification processes and the concomitant effect of the separation on peptide bioactivity.
Peptides are distinctive biomacromolecules that demonstrate potential cytotoxicity and diversified bioactivities against a variety of microorganisms including bacteria, mycobacteria, and fungi via their unique mechanisms of action. Among broad-ranging pharmacologically active peptides, natural marine-originated thiazole-based oligopeptides possess peculiar structural features along with a wide spectrum of exceptional and potent bioproperties. Because of their complex nature and size divergence, thiazole-based peptides (TBPs) bestow a pivotal chemical platform in drug discovery processes to generate competent scaffolds for regulating allosteric binding sites and peptide-peptide interactions. The present study dissertates on the natural reservoirs and exclusive structural components of marine-originated TBPs, with a special focus on their most pertinent pharmacological profiles, which may impart vital resources for the development of novel peptide-based therapeutic agents.
Antimicrobial peptides (AMPs), the self-defence products of organisms, are extensively distributed in plants. They can be classified into several groups, including thionins, defensins, snakins, lipid transfer proteins, glycine-rich proteins, cyclotides and hevein-type proteins. AMPs can be extracted and isolated from different plants and plant organs such as stems, roots, seeds, flowers and leaves. They perform various physiological defensive mechanisms to eliminate viruses, bacteria, fungi and parasites, and so could be used as therapeutic and preservative agents. Research on AMPs has sought to obtain more detailed and reliable information regarding the selection of suitable plant sources and the use of appropriate isolation and purification techniques, as well as examining the mode of action of these peptides. Well-established AMP purification techniques currently used include salt precipitation methods, absorption-desorption, a combination of ion-exchange and reversed-phase C18 solid phase extraction, reversed-phase high-performance liquid chromatography (RP-HPLC), and the sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) method. Beyond these traditional methods, this review aims to highlight new and different approaches to the selection, characterisation, isolation, purification, mode of action and bioactivity assessment of a range of AMPs collected from plant sources. The information gathered will be helpful in the search for novel AMPs distributed in the plant kingdom, as well as providing future directions for the further investigation of AMPs for possible use on humans.
The search for the mechanism of ribosomal peptide bond formation is still ongoing. Even though the actual mechanism of peptide bod formation is still unknown, the dominance of proton transfer in this reaction is known for certain. Therefore, it is vital to take the quantum mechanical effects on proton transfer reaction into consideration; the effects of which were neglected in all previous studies. In this study, we have taken such effects into consideration using a semi-classical approach to the overall reaction mechanism. The M06-2X density functional with the 6-31++G(d,p) basis set was used to calculate the energies of the critical points on the potential energy surface of the reaction mechanism, which are then used in transition state theory to calculate the classical reaction rate. The tunnelling contribution is then added to the classical part by calculating the transmission permeability and tunnelling constant of the reaction barrier, using the numerical integration over the Boltzmann distribution for the symmetrical Eckart potential. The results of this study, which accounts for quantum effects, indicates that the A2451 ribosomal residue induces proton tunnelling in a stepwise peptide bond formation.
Inflammatory conditions are frequently accompanied by increased levels of active proteases, and there is rising interest in methods for their detection to monitor inflammation in a point of care setting. In this work, new sensor materials for disposable single-step protease biosensors based on poly(2-oxazoline) hydrogels cross-linked with a protease-specific cleavable peptide are described. The performance of the sensor material was assessed targeting the detection of matrix metalloproteinase-9 (MMP-9), a protease that has been shown to be an indicator of inflammation in multiple sclerosis and other inflammatory conditions. Films of the hydrogel were formed on gold-coated quartz crystals using thiol-ene click chemistry, and the cross-link density was optimized. The degradation rate of the hydrogel was monitored using a quartz crystal microbalance (QCM) and showed a strong dependence on the MMP-9 concentration. A concentration range of 0-160 nM of MMP-9 was investigated, and a lower limit of detection of 10 nM MMP-9 was determined.
Actinidin was used to pretreat the bovine hide and ultrasonic wave (53 kHz and 500 W) was used for the time durations of 2, 4 and 6 h at 60 °C to extract gelatin samples (UA2, UA4 and UA6, respectively). Control (UAC) gelatin was extracted using ultrasound for 6 h at 60 °C without enzyme pretreatment. There was significant (p < 0.05) increase in gelatin yield as the time duration of ultrasound treatment increased with UA6 giving the highest yield of 19.65%. Gel strength and viscosity of UAC and UA6 extracted gelatin samples were 627.53 and 502.16 g and 16.33 and 15.60 mPa.s, respectively. Longer duration of ultrasound treatment increased amino acids content of the extracted gelatin and UAC exhibited the highest content of amino acids. Progressive degradation of polypeptide chains was observed in the protein pattern of the extracted gelatin as the time duration of ultrasound extraction increased. Fourier transform infrared (FTIR) spectroscopy depicted loss of molecular order and degradation in UA6. Scanning electron microscopy (SEM) revealed protein aggregation and network formation in the gelatin samples with increasing time of ultrasound treatment. The study indicated that ultrasound assisted gelatin extraction using actinidin exhibited high yield with good quality gelatin.
In this work, we proposed a biosensor for trypsin proteolytic activity assay using immobilization of model peptides on screen-printed electrodes (SPE) modified with gold nanoparticles (AuNPs) prepared by electrosynthetic method. Sensing of proteolytic activity was based on electrochemical oxidation of tyrosine residues of peptides. We designed peptides containing N-terminal cysteine residue for immobilization on an SPE, modified with gold nanoparticles, trypsin-specific cleavage site and tyrosine residue as a redox label. The peptides were immobilized on SPE by formation of chemical bonds between mercapto groups of the N-terminal cysteine residues and AuNPs. After the incubation with trypsin, time-dependent cleavage of the immobilized peptides was observed by decline in tyrosine electrochemical oxidation signal. The kinetic parameters of trypsin, such as the catalytic constant (kcat), the Michaelis constant (KM) and the catalytic efficiency (kcat/KM), toward the CGGGRYR peptide were determined as 0.33 ± 0.01 min-1, 198 ± 24 nM and 0.0016 min-1 nM-1, respectively. Using the developed biosensor, we demonstrated the possibility of analysis of trypsin specificity toward the peptides with amino acid residues disrupting proteolysis. Further, we designed the peptides with proline or glutamic acid residues after the cleavage site (CGGRPYR and CGGREYR), and trypsin had reduced activity toward both of them according to the existing knowledge of the enzyme specificity. The developed biosensor system allows one to perform a comparative analysis of the protease steady-state kinetic parameters and specificity toward model peptides with different amino acid sequences.
Amyloidosis is a deleterious condition caused by abnormal amyloid fibril build-up in living tissues. To date, 42 proteins that are linked to amyloid fibrils have been discovered. Amyloid fibril structure variation can affect the severity, progression rate, or clinical symptoms of amyloidosis. Since amyloid fibril build-up is the primary pathological basis for various neurodegenerative illnesses, characterization of these deadly proteins, particularly utilising optical techniques have been a focus. Spectroscopy techniques provide significant non-invasive platforms for the investigation of the structure and conformation of amyloid fibrils, offering a wide spectrum of analyses ranging from nanometric to micrometric size scales. Even though this area of study has been intensively explored, there still remain aspects of amyloid fibrillization that are not fully known, a matter hindering progress in treating and curing amyloidosis. This review aims to provide recent updates and comprehensive information on optical techniques for metabolic and proteomic characterization of β-pleated amyloid fibrils found in human tissue with thorough literature analysis of publications. Raman spectroscopy and SAXS are well established experimental methods for study of structural properties of biomaterials. With suitable models, they offer extended information for valid proteomic analysis under physiologically relevant conditions. This review points to evidence that despite limitations, these techniques are able to provide for the necessary output and proteomics indication in order to extrapolate the aetiology of amyloid fibrils for reliable diagnostic purposes. Our metabolic database may also contribute to elucidating the nature and function of the amyloid proteome in development and clearance of amyloid diseases.
The ultra-high-performance liquid chromatography tandem mass spectrometry (UHPLC-MS) method was built to quantify the casein glycomacropeptide (CGMP) in bovine dairy products accurately based on targeted proteomics. Qualitative analysis of theoretical peptides was carried out using high-resolution mass spectrometry (HRMS) and protein software. Isotope-labeled characteristic peptides were acquired via the labeled amino acid condensation method to correct the matrix effects. Peptide MAIPPK was the representative characteristic peptide for distinguishing the CGMP from κ-casein through trypsin digestion. After optimizing the pre-treatment conditions, the final 8% oxidant concentration was selected and the 10% formic acid concentration with 2.5 h oxidation time. Moreover, the results of methodological verification showed that the recovery rate was 103.7%, meanwhile the precision of inter-day and intra-day was less than 5%. In conclusion, the research demonstrated the characteristic peptide MAIPPK could quantitatively applied to detect CGMP in dairy products.