Design Of Peptides Research Articles

Computational design of anti-cancer peptides tailored to target specific tumor markers

Anti-cancer peptides (ACPs) are short peptides known for their ability to inhibit tumor cell proliferation, migration, and the formation of tumor blood vessels. In this study, we designed ACPs to target receptors often overexpressed in cancer using a systematic in silico approach. Three target receptors (CXCR1, DcR3, and OPG) were selected for their significant roles in cancer pathogenesis and tumor cell proliferation. Our peptide design strategy involved identifying interacting residues (IR) of these receptors, with their natural ligands serving as a reference for designing peptides specific to each receptor. The natural ligands of these receptors, including IL8 for CXCR1, TL1A for DcR3, and RANKL for OPG, were identified from the literature. Using the identified interacting residues (IR), we generated a peptide library through simple permutation and predicted the structure of each peptide. All peptides were analyzed using the web-based prediction server for Anticancer peptides, AntiCP. Docking simulations were then conducted to analyze the binding efficiencies of peptides with their respective target receptors, using VEGA ZZ and Chimera for interaction analysis. Our analysis identified HPKFIKELR as the interacting residues (IR) of CXCR-IL8. For DcR3, we utilized three domains from TL1A (TDSYPEP, TKEDKTF, LGLAFTK) as templates, along with two regions (SIKIPSS and PDQDATYP) from RANKL, to generate a library of peptide analogs. Subsequently, peptides for each receptor were shortlisted based on their predicted anticancer properties as determined by AntiCP and were subjected to docking analysis. After docking, peptides that exhibited the least binding energy were further analyzed for their detailed interaction with their respective receptors. Among these, peptides C9 (HPKFELY) and C7 (HPKFEWL) for CXCR1, peptides D6 (ADSYPQP) and D18 (AFSYPFP) for DcR3, and peptides P19 (PDTYPQDP) and p16 (PDQDATYP) for OPG, demonstrated the highest affinity and stronger interactions compared to the other peptides. Although in silico predictions indicated a favorable binding affinity of the designed peptides with target receptors, further experimental validation is essential to confirm their binding affinity, stability and pharmacokinetic characteristics.

Design of target specific peptide inhibitors using generative deep learning and molecular dynamics simulations

We introduce a computational approach for the design of target-specific peptides. Our method integrates a Gated Recurrent Unit-based Variational Autoencoder with Rosetta FlexPepDock for peptide sequence generation and binding affinity assessment. Subsequently, molecular dynamics simulations are employed to narrow down the selection of peptides for experimental assays. We apply this computational strategy to design peptide inhibitors that specifically target β-catenin and NF-κB essential modulator. Among the twelve β-catenin inhibitors, six exhibit improved binding affinity compared to the parent peptide. Notably, the best C-terminal peptide binds β-catenin with an IC50 of 0.010 ± 0.06 μM, which is 15-fold better than the parent peptide. For NF-κB essential modulator, two of the four tested peptides display substantially enhanced binding compared to the parent peptide. Collectively, this study underscores the successful integration of deep learning and structure-based modeling and simulation for target specific peptide design.

Structure-Guided Discovery of PD-1/PD-L1 Interaction Inhibitors: Peptide Design, Screening, and Optimization via Computation-Aided Phage Display Engineering.

Cancer immunotherapy harnesses the immune system to combat tumors and has emerged as a major cancer treatment modality. The PD-1/PD-L1 immune checkpoint modulates interactions between tumor cells and T cells and has been extensively targeted in cancer immunotherapy. However, the monoclonal antibodies known to target this immune checkpoint have considerable side effects, and novel PD-1/PD-L1 inhibitors are therefore required. Herein, a peptide inhibitor to disrupt PD-1/PD-L1 interactions was designed through structure-driven phage display engineering coupled to computational modification and optimization. BetaPb, a novel peptide library constructed by using the known structure of PD-1/PD-L, was used to develop inhibitors against the immune checkpoint, and specific peptides with high affinity toward PD-1 were screened through enzyme-linked immunosorbent assays, homogeneous time-resolved fluorescence, and biolayer interferometry. A potential inhibitor, B8, was preliminarily screened through biopanning. The binding affinity of B8 toward PD-1 was confirmed through computation-aided optimization. Assessment of B8 variants (B8.1, B8.2, B8.3, B8.4, and B8.5) demonstrated their attenuation of PD-1/PD-L1 interactions. B8.4 exhibited the strongest attenuation efficiency at a half-maximal effective concentration of 0.1 μM and the strongest binding affinity to PD-1 (equilibrium dissociation constant = 0.1 μM). B8.4 outperformed the known PD-1/PD-L1 interaction inhibitor PL120131 in disrupting PD-1/PD-L1 interactions, revealing that B8.4 has remarkable potential for modification to yield an antitumor agent. This study provides valuable information for the future development of peptide-based drugs, therapeutics, and immunotherapies for cancer.

Hybrid 2D/3D-quantitative structure–activity relationship studies on the bioactivities and molecular mechanism of antibacterial peptides

Antimicrobial peptide (AMP) is the polypeptide, which protects the organism avoiding attack from pathogenic bacteria. Studies have shown that there were some antimicrobial peptides with molecular action mechanism involved in crossing the cell membrane without inducing severe membrane collapse, then interacting with cytoplasmic target-nucleic acid, and exerting antibacterial activity by interfacing the transmission of genetic information of pathogenic microorganisms. However, the relationship between the antibacterial activities and peptide structures was still unclear. Therefore, in the present work, a series of AMPs with a sequence of 20 amino acids was extracted from DBAASP database, then, quantitative structure–activity relationship (QSAR) methods were conducted on these peptides. In addition, novel antimicrobial peptides with stronger antimicrobial activities were designed according to the information originated from the constructed models. Hence, the outcome of this study would lay a solid foundation for the in-silico design and exploration of novel antibacterial peptides with improved activity activities.

Design of Functional Globular β-Sheet Miniproteins.

The design of discrete β-sheet peptides is far less advanced than e. g. the design of α-helical peptides. The reputation of β-sheet peptides as being poorly soluble and aggregation-prone often hinders active design efforts. Here, we show that this reputation is unfounded. We demonstrate this by looking at the β-hairpin and WW domain. Their structure and folding have been extensively studied and they have long served as model systems to investigate protein folding and folding kinetics. The resulting fundamental understanding has led to the development of hyperstable β-sheet scaffolds that fold at temperatures of 100 °C or high concentrations of denaturants. These have been used to design functional miniproteins with protein or nucleic acid binding properties, in some cases with such success that medical applications are conceivable. The β-sheet scaffolds are not always completely rigid, but can be specifically designed to respond to changes in pH, redox potential or presence of metal ions. Some engineered β-sheet peptides also exhibit catalytic properties, although not comparable to those of natural proteins. Previous reviews have focused on the design of stably folded and non-aggregating β-sheet sequences. In our review, we now also address design strategies to obtain functional miniproteins from β-sheet folding motifs.

Modular design of cyclic peptide – polymer conjugate nanotubes for delivery and tunable release of anti-cancer drug compounds

High aspect-ratio nanomaterials have recently emerged as promising drug delivery vehicles due to evidence of strong cellular association and prolonged in vivo circulation times. Cyclic peptide - polymer conjugate nanotubes are excellent candidates due to their elongated morphology, their supramolecular composition and high degree of pliability due to the versatility in manipulating amino acid sequence and polymer type. In this work, we explore the use of a nanotube structure on which a potent anti-cancer drug, camptothecin, is attached alongside hydrophilic or amphiphilic RAFT polymers, which shield the cargo. We show that subtle modifications to the cleavable linker type and polymer architecture have a dramatic influence over the rate of drug release in biological conditions. In vitro studies revealed that multiple cancer cell lines in 2D and 3D models responded effectively to the nanotube treatment, and analogous fluorescently labelled materials revealed key mechanistic information regarding the degree of cellular uptake and intracellular fate. Importantly, the ability to instruct specific drug release profiles indicates a potential for these nanomaterials as vectors which can provide sustained drug concentrations for a maximal therapeutic effect.

A rationally designed antimicrobial peptide from structural and functional insights of Clostridioides difficile translation initiation factor 1.

A significant increase of hospital-acquired bacterial infections during the COVID-19 pandemic has become an urgent medical problem. Clostridioides difficile is an urgent antibiotic-resistant bacterial pathogen and a leading causative agent of nosocomial infections. The increasing recurrence of C. difficile infection and antibiotic resistance in C. difficile has led to an unmet need for the discovery of new compounds distinctly different from present antimicrobials, while antimicrobial peptides as promising alternatives to conventional antibiotics have attracted growing interest recently. Protein synthesis is an essential metabolic process in all bacteria and a validated antibiotic target. Initiation factor 1 from C. difficile (Cd-IF1) is the smallest of the three initiation factors that acts to establish the 30S initiation complex to initiate translation during protein biosynthesis. Here, we report the solution nuclear magnetic resonance (NMR) structure of Cd-IF1 which adopts a typical β-barrel fold and consists of a five-stranded β-sheet and one short α-helix arranged in the sequential order β1-β2-β3-α1-β4-β5. The interaction of Cd-IF1 with the 30S ribosomal subunit was studied by NMR titration for the construction of a structural model of Cd-IF1 binding with the 30S subunit. The short α-helix in IF1 was found to be critical for IF1 ribosomal binding. A peptide derived from this α-helix was tested and displayed a high ability to inhibit the growth of C. difficile and other bacterial strains. These results provide a clue for the rational design of new antimicrobials.IMPORTANCEBacterial infections continue to represent a major worldwide health hazard due to the emergence of drug-resistant strains. Clostridioides difficile is a common nosocomial pathogen and the causative agent in many infections resulting in an increase in morbidity and mortality. Bacterial protein synthesis is an essential metabolic process and an important target for antibiotic development; however, the precise structural mechanism underlying the process in C. difficile remains unknown. This study reports the solution structure of C. difficile translation initiation factor 1 (IF1) and its interaction with the 30S ribosomal subunit. A short α-helix in IF1 structure was identified as critically important for ribosomal binding and function in regulating the translation initiation, which allowed a rational design of a new peptide. The peptide demonstrated a high ability to inhibit bacterial growth with broad-spectrum antibacterial activity. This study provides a new clue for the rational design of new antimicrobials against bacterial infections.

Rational Design of a Potent Antimicrobial Peptide Based on the Active Region of a Gecko Cathelicidin.

The emergence of multidrug-resistant (MDR) bacteria presents a significant challenge to public health, increasing the risk of infections that are resistant to current antibiotic treatment. Antimicrobial peptides (AMPs) offer a promising alternative to conventional antibiotics in the prevention of MDR bacterial infections. In the present study, we identified a novel cathelicidin AMP from Gekko japonicus, which exhibited broad-spectrum antibacterial activity against both Gram-negative and Gram-positive bacteria, with minimal inhibitory concentrations ranging from 2.34 to 4.69 μg/mL. To improve its potential therapeutic application, a series of peptides was synthesized based on the active region of the gecko-derived cathelicidin. The lead peptide (RH-16) showed an antimicrobial activity comparable to that of the parent peptide. Structural characterization revealed that RH-16 adopted an amphipathic α-helical conformation. Furthermore, RH-16 demonstrated neither hemolytic nor cytotoxic activity but effectively killed a wide range of clinically isolated, drug-resistant bacteria. The antimicrobial activity of RH-16 was attributed to the nonspecific targeting of bacterial membranes, leading to rapid bacterial membrane permeabilization and rupture. RH-16 also retained its antibacterial activity in plasma and exhibited mild toxicity in vivo. Notably, RH-16 offered robust protection against skin infection in a murine model. Therefore, this newly identified cathelicidin AMP may be a strong candidate for future pharmacological development targeting multidrug resistance. The use of a rational design approach for isolating the minimal antimicrobial unit may accelerate the transition of natural AMPs to clinically applicable antibacterial agents.

Enantiomer-Dependent Supramolecular Immunosuppressive Modulation for Tissue Reconstruction.

Modulating the properties of biomaterials in terms of the host immune response is critical for tissue repair and regeneration. However, it is unclear how the preference for the cellular microenvironment manipulates the chiral immune responses under physiological or pathological conditions. Here, we reported that in vivo and in vitro oligopeptide immunosuppressive modulation was achieved by manipulation of macrophage polarization using chiral tetrapeptide (Ac-FFFK-OH, marked as FFFK) supramolecular polymers. The results suggested that chiral FFFK nanofibers can serve as a defense mechanism in the restoration of tissue homeostasis by upregulating macrophage M2 polarization via the Src-STAT6 axis. More importantly, transiently acting STAT6, insufficient to induce a sustained polarization program, then passes the baton to EGR2, thereby continuously maintaining the M2 polarization program. It is worth noting that the L-chirality exhibits a more potent effect in inducing macrophage M2 polarization than does the D-chirality, leading to enhanced tissue reconstruction. These findings elucidate the crucial molecular signals that mediate chirality-dependent supramolecular immunosuppression in damaged tissues while also providing an effective chiral supramolecular strategy for regulating macrophage M2 polarization and promoting tissue injury repair based on the self-assembling chiral peptide design.

De novo design of biocatalytic and thermostable heterochiral peptide as a hydrolase model

De novo design of biocatalytic and thermostable heterochiral peptide as a hydrolase model

Furthering design of mimetic lipoprotein peptides through all-atom simulations

Furthering design of mimetic lipoprotein peptides through all-atom simulations

Rational Design of Amphipathic Antimicrobial Peptides with Alternating L-/D-Amino Acids That Form Helical Structures.

Antimicrobial peptides (AMPs) are promising therapeutic agents against bacteria. We have previously reported an amphipathic AMP Stripe composed of cationic L-Lys and hydrophobic L-Leu/L-Ala residues, and Stripe exhibited potent antimicrobial activity against Gram-positive and Gram-negative bacteria. Gramicidin A (GA), composed of repeating sequences of L- and D-amino acids, has a unique β6.3-helix structure and exhibits broad antimicrobial activity. Inspired by the structural properties and antimicrobial activities of LD-alternating peptides such as GA, in this study, we designed Stripe derivatives with LD-alternating sequences. We found that simply alternating L- and D-amino acids in the Stripe sequence to give StripeLD caused a reduction in antimicrobial activity. In contrast, AltStripeLD, with cationic and hydrophobic amino acids rearranged to yield an amphipathic distribution when the peptide adopts a β6.3-helix, displayed higher antimicrobial activity than AltStripe. These results suggest that alternating L-/D-cationic and L-/D-hydrophobic amino acids in accordance with the helical structure of an AMP may be a useful way to improve antimicrobial activity and develop new AMP drugs.

Design of an Artificial Peptide Inspired by Transmembrane Mitochondrial Protein for Escorting Exogenous DNA into the Mitochondria to Restore their Functions by Simultaneous Multiple Gene Expression (Adv. Funct. Mater. 8/2024)

Design of an Artificial Peptide Inspired by Transmembrane Mitochondrial Protein for Escorting Exogenous DNA into the Mitochondria to Restore their Functions by Simultaneous Multiple Gene Expression (Adv. Funct. Mater. 8/2024)

Generative β-hairpin design using a residue-based physicochemical property landscape

De novo peptide design is a new frontier that has broad application potential in the biological and biomedical fields. Most existing models for de novo peptide design are largely based on sequence homology that can be restricted based on evolutionarily derived protein sequences and lack the physicochemical context essential in protein folding. Generative machine learning for de novo peptide design is a promising way to synthesize theoretical data that are based on, but unique from, the observable universe. In this study, we created and tested a custom peptide generative adversarial network intended to design peptide sequences that can fold into the β-hairpin secondary structure. This deep neural network model is designed to establish a preliminary foundation of the generative approach based on physicochemical and conformational properties of 20 canonical amino acids, for example, hydrophobicity and residue volume, using extant structure-specific sequence data from the PDB. The beta generative adversarial network model robustly distinguishes secondary structures of β hairpin from α helix and intrinsically disordered peptides with an accuracy of up to 96% and generates artificial β-hairpin peptide sequences with minimum sequence identities around 31% and 50% when compared against the current NCBI PDB and nonredundant databases, respectively. These results highlight the potential of generative models specifically anchored by physicochemical and conformational property features of amino acids to expand the sequence-to-structure landscape of proteins beyond evolutionary limits.

Critical Aggregation Concentration Can be a Predictor of Doxorubicin Delivery Performance of Self-Assembling Amphiphilic Peptides with Different Hydrophobic Tails

Amphiphilic peptides hold great potential as drug delivery systems. A popular peptide design approach has been to place amino acids in the peptide sequence based on their known properties. On the other hand, the directed discovery approach aims to screen a sequence space for a desired property. However, screening amphiphilic peptides for desirable drug delivery properties is not possible without a quantity that is predictive of these properties. We studied the predictive power of critical aggregation concentration (CAC) values on the drug delivery performance of a series of amphiphilic peptides with different hydrophobic tails and close CAC values. The CAC values were predicted by our previously developed model and doxorubicin was used as a model hydrophobic drug. All peptides showed close drug loading, entrapment efficiency, and release profile. They also formed similar spherical particles by assembling in reverse β-sheet arrangements regardless of drug presence. Moreover, the assembled particles were able to accumulate doxorubicin inside ordinary as well as drug-resistant breast cancer cells and enhance its toxicity up to 39 and 17 folds, respectively. It can be concluded that similar drug delivery properties displayed by the peptides can be attributed to their similar hydrophilic-lipophilic balance as reflected in their close CAC values.

Rational Design of Antifungal Peptides Based on the γ-Core Motif of a Neosartorya (Aspergillus) fischeri Antifungal Protein to Improve Structural Integrity, Efficacy, and Spectrum.

Antifungal peptides offer promising alternative compounds for the treatment of fungal infections, for which new antifungal compounds are urgently needed. Constant and broad antifungal spectra of these peptides play essential roles in their reliable therapeutic application. It has been observed that rationally designed peptides using the evolutionarily conserved γ-core region (GXC-X3-9-C) of an antifungal protein from Neosartorya (Aspergillus) fischeri highly inhibit the growth of fungi. The cysteines in these peptides have free sulfhydryl groups, which allow cyclization and dimerization under oxidative conditions, thereby impairing antifungal efficacy. To overcome this problem, one or two cysteine residues were substituted by serines or S-tert-butyl was applied as a cysteine-protecting group. Furthermore, structural integrity and antifungal efficacy investigations before and after oxidative exposure revealed that substituting both cysteines with serines and S-tert-butylation helped maintain the structural integrity. However, it slightly decreased the antifungal efficacy against a yeast, Candida albicans. Interestingly, S-tert-butylation maintained the efficacy and could extend the antifungal activity to a mold, Aspergillus fumigatus. Usually, cyclization and dimerization did not influence the antifungal efficacy of most peptides. Additionally, hemolysis tests and Galleria mellonella toxicity model experiments indicated that none of the applied modifications made the peptides harmful to animals.

Recombinant Antibody Fragments for Immunotherapy of Parkinson's Disease.

Parkinson's disease (PD) is the second most common age-related neurodegenerative disorder. Multiple genetic and environmental factors leading to progressive loss of dopaminergic neurons in the substantia nigra pars compacta (SN) and consequent depletion of dopamine were described. Current clinical approaches, such as dopamine replacement or deep brain stimulation using surgically implanted probes, provide symptomatic relief but cannot modify disease progression. Therefore, disease-modifying therapeutic tools are urgently needed. Immunotherapy approaches, including passive transfer of protective antibodies and their fragments, have shown therapeutic efficacy in several animal models of neurodegenerative diseases, including PD. Recombinant antibody fragments are promising alternatives to conventional full-length antibodies. Modern computational approaches and molecular biology tools, directed evolution methodology, and the design of tissue-penetrating fusion peptides allowed for the development of recombinant antibody fragments with superior specificity and affinity, reduced immunogenicity, the capacity to target hidden epitopes and cross the blood-brain barrier (BBB), higher solubility and stability, the ability to refold after heat denaturation, and inexpensive large-scale production. In addition, antibody fragments do not induce microglia Fcγ receptor (FcγR)-mediated proinflammatory response and tissue damage in the central nervous system (CNS), because they lack the Fc portion of the immunoglobulin molecule. In the present review, we summarized data on recombinant antibody fragments evaluated as immunotherapeutics in preclinical models of PD and discussed their potential for developing therapeutic and preventive protocols for patients with PD.

Design of a novel analogue peptide with potent antibiofilm activities against Staphylococcus aureus based upon a sapecin B-derived peptide

Nowadays, antimicrobial peptides are promising to confront the existing global crisis of antibiotic resistance. Here, a novel analogue peptide (mKLK) was designed based upon a D-form amidated sapecin B-derived peptide (KLK) by replacing two lysine residues with two tryptophan and one leucine by lysine, and inserting one alanine. The mKLK displayed superior amphipathic helixes in which the most of hydrophobic residues are confined to one face of the helix and had a higher hydrophobic moment compared with KLK. The mKLK retained its antibacterial activity and structure in human serum, suggesting its stability to proteolytic degradation. The values of MIC and MBC for mKLK were equal to those of KLK against clinical strains of methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-susceptible Staphylococcus aureus (MSSA). However, mKLK showed more capability of in vitro inhibiting, eradicating, and dispersing MRSA and MSSA biofilms compared with KLK. Furthermore, a remarkable inhibitory activity of mKLK against MRSA and MSSA biofilms was seen in the murine model of catheter-associated biofilm infection. Results of this study show that mKLK not only exhibits antibacterial activity and serum stability but also a potent biofilm inhibitory activity at sub-MIC concentrations, confirming its potential therapeutic advantage for preventing biofilm-associated MRSA and MSSA infections.

Functionalization of Bacterial Cellulose with the Antimicrobial Peptide KR-12 via Chimerical Cellulose-Binding Peptides.

Bacterial-derived cellulose (BC) has been studied as a promising material for biomedical applications, including wound care, due to its biocompatibility, water-holding capacity, liquid/gas permeability, and handleability properties. Although BC has been studied as a dressing material for cutaneous wounds, to date, BC inherently lacks antibacterial properties. The current research utilizes bifunctional chimeric peptides containing carbohydrate binding peptides (CBP; either a short version or a long version) and an antimicrobial peptide (AMP), KR-12. The secondary structure of the chimeric peptides was evaluated and confirmed that the α-helix structure of KR-12 was retained for both chimeric peptides evaluated (Long-CBP-KR12 and Short-CBP-KR12). Chimeric peptides and their individual components were assessed for cytotoxicity, where only higher concentrations of Short-CBP and longer timepoints of Short-CBP-KR12 exposure exhibited negative effects on metabolic activity, which was attributed to solubility issues. All KR-12-containing peptides exhibited antibacterial activity in solution against Escherichia coli (E. coli) and Pseudomonas aeruginosa (P. aeruginosa). The lipopolysaccharide (LPS) binding capability of the peptides was evaluated and the Short-CBP-KR12 peptide exhibited enhanced LPS-binding capabilities compared to KR-12 alone. Both chimeric peptides were able to bind to BC and were observed to be retained on the surface over a 7-day period. All functionalized materials exhibited no adverse effects on the metabolic activity of both normal human dermal fibroblasts (NHDFs) and human epidermal keratinocyte (HaCaT) epithelial cells. Additionally, the BC tethered chimeric peptides exhibited antibacterial activity against E. coli. Overall, this research outlines the design and evaluation of chimeric CBP-KR12 peptides for developing antimicrobial BC membranes with potential applications in wound care.

Design of dual peptide-conjugated hydrogels for proliferation and differentiation of human pluripotent stem cells

Completely synthetic cell cultivation materials for human pluripotent stem cells (hPSCs) are important for the future clinical use of hPSC-derived cells. Currently, cell culture materials conjugated with extracellular matrix (ECM)-derived peptides are being prepared using only one specific integrin-targeting peptide. We designed dual peptide-conjugated hydrogels, for which each peptide was selected from different ECM sites: the laminin β4 chain and fibronectin or vitronectin, which can target α6β1 and α2β1 or αVβ5. hPSCs cultured on dual peptide-conjugated hydrogels, especially on hydrogels conjugated with peptides obtained from the laminin β4 chain and vitronectin with a low peptide concentration of 200 μg/mL, showed high proliferation ability over the long term and differentiated into cells originating from 3 germ layers in vivo as well as a specific lineage of cardiac cells. The design of grafting peptides was also important, for which a joint segment and positive amino acids were added into the designed peptide. Because of the designed peptides on the hydrogels, only 200 μg/mL peptide solution was sufficient for grafting on the hydrogels, and the hydrogels supported hPSC cultures long-term; in contrast, in previous studies, greater than 1000 μg/mL peptide solution was needed for the grafting of peptides on cell culture materials.