bims-cepepe 2023-09-10 papers

bims-cepepe

Biomed News

on Cell-penetrating peptides

Issue of 2023–09–10
fiveteen papers selected by
Henry Lamb, Queensland University of Technology

Design of Cyclic Peptides Targeting Protein-Protein Interactions Using AlphaFold.
Computational Evaluation of Peptide-Protein Binding Affinities: Application of Potential of Mean Force Calculations to SH2 Domains.
Assessing the fast prediction of peptide conformers and the impact of non-natural modifications.
Structural dynamics influences the antibacterial activity of a cell-penetrating peptide (KFF)3K.
Cell-penetrating peptides predictors: A comparative analysis of methods and datasets.
Defect Formation and Peptide Permeation across Phospholipid Membranes.
Cellular uptake and trafficking of peptide-based drug delivery systems for miRNA.
Designing Cyclic-Constrained Peptides to Inhibit Human Phosphoglycerate Dehydrogenase.
Trifluoromethylthiolation of Tryptophan and Tyrosine Derivatives: A Tool for Enhancing the Local Hydrophobicity of Peptides.
Synthesis and Biochemical Evaluation of Monocarboxylic GRB2 SH2 Domain Inhibitors.
Effect of Spacer Length Modification of the Cationic Side Chain on the Energetics of Antimicrobial Peptide Binding to Membrane-Mimetic Bilayers.
PepAnalyzer: predicting peptide properties using its sequence.
Peptides Targeting HER2-Positive Breast Cancer Cells and Applications in Tumor Imaging and Delivery of Chemotherapeutics.
Discovery of a potent, orally available tricyclic derivative as a novel BRD4 inhibitor for melanoma.
Peptides as Baits for the Coprecipitation of SH2 Domain-Containing Proteins.

Int J Mol Sci. 2023 Aug 26. pii: 13257. [Epub ahead of print]24(17):

Design of Cyclic Peptides Targeting Protein-Protein Interactions Using AlphaFold.

Takatsugu Kosugi, Masahito Ohue.

  More than 930,000 protein-protein interactions (PPIs) have been identified in recent years, but their physicochemical properties differ from conventional drug targets, complicating the use of conventional small molecules as modalities. Cyclic peptides are a promising modality for targeting PPIs, but it is difficult to predict the structure of a target protein-cyclic peptide complex or to design a cyclic peptide sequence that binds to the target protein using computational methods. Recently, AlphaFold with a cyclic offset has enabled predicting the structure of cyclic peptides, thereby enabling de novo cyclic peptide designs. We developed a cyclic peptide complex offset to enable the structural prediction of target proteins and cyclic peptide complexes and found AlphaFold2 with a cyclic peptide complex offset can predict structures with high accuracy. We also applied the cyclic peptide complex offset to the binder hallucination protocol of AfDesign, a de novo protein design method using AlphaFold, and we could design a high predicted local-distance difference test and lower separated binding energy per unit interface area than the native MDM2/p53 structure. Furthermore, the method was applied to 12 other protein-peptide complexes and one protein-protein complex. Our approach shows that it is possible to design putative cyclic peptide sequences targeting PPI.

Keywords:  AfDesign; AlphaFold; cyclic peptide; peptide design; protein–protein interaction (PPI)

DOI:  https://doi.org/10.3390/ijms241713257
Methods Mol Biol. 2023 ;2705 113-133

Computational Evaluation of Peptide-Protein Binding Affinities: Application of Potential of Mean Force Calculations to SH2 Domains.

Paolo Calligari, Lorenzo Stella, Gianfranco Bocchinfuso.

  Many biological functions are mediated by protein-protein interactions (PPIs), often involving specific structural modules, such as SH2 domains. Inhibition of PPIs is a pharmaceutical strategy of growing importance. However, a major challenge in the design of PPI inhibitors is the large interface involved in these interactions, which, in many cases, makes inhibition by small organic molecules ineffective. Peptides, which cover a wide range of dimensions and can be opportunely designed to mimic protein sequences at PPI interfaces, represent a valuable alternative to small molecules. Computational techniques able to predict the binding affinity of peptides for the target domain or protein represent a crucial stage in the workflow for the design of peptide-based drugs. This chapter describes a protocol to obtain the potential of mean force (PMF) for peptide-SH2 domain binding, starting from umbrella sampling (US) molecular dynamics (MD) simulations. The PMF profiles can be effectively used to predict the relative standard binding free energies of different peptide sequences.

Keywords:  Molecular dynamics simulations; Peptide design; Potential of mean force; Protein–protein interactions; Relative standard binding free energy; Umbrella sampling

DOI:  https://doi.org/10.1007/978-1-0716-3393-9_7
J Mol Graph Model. 2023 Aug 22. pii: S1093-3263(23)00206-1. [Epub ahead of print]125 108608

Assessing the fast prediction of peptide conformers and the impact of non-natural modifications.

Rodrigo Ochoa, Thomas Fox.

  We present an assessment of different approaches to predict peptide structures using modeling tools. Several small molecule, protein, and peptide-focused methodologies were used for the fast prediction of conformers for peptides shorter than 30 amino acids. We assessed the effect of including restraints based on annotated or predicted secondary structure motifs. A number of peptides in bound conformations and in solution were collected to compare the tools. In addition, we studied the impact of changing single amino acids to non-natural residues using molecular dynamics simulations. Deep learning methods such as AlphaFold2, or the combination of physics-based approaches with secondary structure information, produce the most accurate results for natural sequences. In the case of peptides with non-natural modifications, modeling the peptide containing natural amino acids first and then modifying and simulating the peptide using benchmarked force fields is a recommended pipeline. The results can guide the modeling of oligopeptides for drug discovery projects.

Keywords:  Conformers; Molecular dynamics simulations; Peptides; Secondary structure prediction; Structural bioinformatics

DOI:  https://doi.org/10.1016/j.jmgm.2023.108608
Sci Rep. 2023 Sep 08. 13(1): 14826

Structural dynamics influences the antibacterial activity of a cell-penetrating peptide (KFF)3K.

Julia Macyszyn, Piotr Chyży, Michał Burmistrz, Małgorzata Lobka, Joanna Miszkiewicz, Monika Wojciechowska, Joanna Trylska.

Given the widespread demand for novel antibacterial agents, we modified a cell-penetrating peptide (KFF)3K to transform it into an antibacterial peptide. Namely, we inserted a hydrocarbon staple into the (KFF)3K sequence to induce and stabilize its membrane-active secondary structure. The staples were introduced at two positions, (KFF)3K[5-9] and (KFF)3K[2-6], to retain the initial amphipathic character of the unstapled peptide. The stapled analogues are protease resistant contrary to (KFF)3K; 90% of the stapled (KFF)3K[5-9] peptide remained undigested after incubation in chymotrypsin solution. The stapled peptides showed antibacterial activity (with minimal inhibitory concentrations in the range of 2-16 µM) against various Gram-positive and Gram-negative strains, contrary to unmodified (KFF)3K, which had no antibacterial effect against any strain at concentrations up to 32 µM. Also, both stapled peptides adopted an α-helical structure in the buffer and micellar environment, contrary to a mostly undefined structure of the unstapled (KFF)3K in the buffer. We found that the antibacterial activity of (KFF)3K analogues is related to their disruptive effect on cell membranes and we showed that by stapling this cell-penetrating peptide, we can induce its antibacterial character.

DOI: https://doi.org/10.1038/s41598-023-38745-y
Mol Inform. 2023 Sep 06. e2300104

Cell-penetrating peptides predictors: A comparative analysis of methods and datasets.

Karen Guerrero-Vázquez, Gabriel Del Rio, Carlos A Brizuela.

  Cell-Penetrating Peptides (CPP) are emerging as an alternative to small-molecule drugs to expand the range of biomolecules that can be targeted for therapeutic purposes. Due to the importance of identifying and designing new CPP, a great variety of predictors have been developed to achieve these goals. To establish a ranking for these predictors, a couple of recent studies compared their performances on specific datasets, yet their conclusions cannot determine if the ranking obtained is due to the model, the set of descriptors or the datasets used to test the predictors. We present a systematic study of the influence of the peptide sequence's similarity of the datasets on the predictors' performance. The analysis reveals that the datasets used for training have a stronger influence on the predictors performance than the model or descriptors employed. We show that datasets with low sequence similarity between the positive and negative examples can be easily separated, and the tested classifiers showed good performance on them. On the other hand, a dataset with high sequence similarity between CPP and non-CPP will be a hard dataset, and it should be the one to be used for assessing the performance of new predictors.

Keywords:  cell-penetrating peptides; datasets; machine learning; peptide sequence similarity

DOI:  https://doi.org/10.1002/minf.202300104
J Phys Chem B. 2023 Sep 07.

Defect Formation and Peptide Permeation across Phospholipid Membranes.

Ron Elber.

Cell penetrating peptides (CPPs) are natural agents that efficiently permeate biological membranes. They are frequently positively charged, which is surprising since membranes pose hydrophobic barriers. In this Perspective, I discuss computations and experiments of a permeation model that couples permeant displacement with a membrane defect. We call the proposed mechanism Defect Assisted by Charge (DAC) and illustrate that it reduces the free energy barrier for translocation. A metastable state at the center of the membrane may be observed due to the charge interactions with the phospholipid head groups at the two leaflets. The combination of experiments and simulations sheds light on the mechanisms of a charged peptide translocation across phospholipid membranes.

DOI: https://doi.org/10.1021/acs.jpcb.3c04895
Eur J Pharm Biopharm. 2023 Sep 02. pii: S0939-6411(23)00232-1. [Epub ahead of print]

Cellular uptake and trafficking of peptide-based drug delivery systems for miRNA.

Ivana Ruseska, Andreas Zimmer.

  Today, macromolecular compounds such as microRNAs (miRNAs) are becoming more and more widespread as leading therapeutics. However, their application is limited mostly due to their poor stability, limited cellular uptake, and poor target specificity. Cell-penetrating peptides (CPPs), a group of positively charged peptides, represent a breakthrough as delivery systems for macromolecules. In the present study, we used two types of nanoparticles which differ in the type of CPP used for their manufacturing. The first type is composed of protamine, an arginine rich CPP, which is highly positively charged. The arginine residues are able to form electrostatic interactions with miRNAs, stabilize them, and deliver them to cells. The second type is composed of the N-Ter peptide (also known as MPG), an amphipathic peptide rich in lysine. The positively charged parts of the N-Ter peptide electrostatically stabilize miRNAs, whereas its amphipathic character allows it to successfully traverse cell membranes. We used miRNA-27a, a negative regulator of adipogenesis, to form nanoparticles with the peptides and traced their uptake in 3T3-L1 preadipocytes. Motivated by the lengthy discourse regarding the uptake mechanism of CPPs, the focus of our study was to analyse and understand the internalization of proticles (protamine nanoparticles) and N-Ter complexes. The nanoparticles were characterized regarding size, size distribution, and zeta potential, and their cytotoxicity was tested in 3T3-L1 cells. The uptake studies were performed by varying the experimental conditions such as time, concentration, and temperature, as well as by applying different inhibitors of endocytosis. Furthermore, we assessed the biological effect of miRNA-27a on the pro-adipogenic machinery. The obtained data have shown that protamine and the N-Ter peptide form positively charged nanoparticles through non-covalent complexation. The uptake of proticles and N-Ter complexes was found to be dependent on time, concentration, and temperature, and different uptake pathways were discovered to be involved in the internalization of the different nanoparticles. Furthermore, both types of nanoparticles induced the anti-adipogenic effect of miRNA-27a, demonstrating that this approach can be used as a novel miRNA replacement therapy in the treatment of obesity and obesity-related disorders.

Keywords:  cell-penetrating peptides; direct translocation; drug delivery systems; endocytosis; miRNA; uptake kinetics

DOI:  https://doi.org/10.1016/j.ejpb.2023.08.019
Molecules. 2023 Sep 04. pii: 6430. [Epub ahead of print]28(17):

Designing Cyclic-Constrained Peptides to Inhibit Human Phosphoglycerate Dehydrogenase.

Xiaoyu Qing, Qian Wang, Hanyu Xu, Pei Liu, Luhua Lai.

  Although loop epitopes at protein-protein binding interfaces often play key roles in mediating oligomer formation and interaction specificity, their binding sites are underexplored as drug targets owing to their high flexibility, relatively few hot spots, and solvent accessibility. Prior attempts to develop molecules that mimic loop epitopes to disrupt protein oligomers have had limited success. In this study, we used structure-based approaches to design and optimize cyclic-constrained peptides based on loop epitopes at the human phosphoglycerate dehydrogenase (PHGDH) dimer interface, which is an obligate homo-dimer with activity strongly dependent on the oligomeric state. The experimental validations showed that these cyclic peptides inhibit PHGDH activity by directly binding to the dimer interface and disrupting the obligate homo-oligomer formation. Our results demonstrate that loop epitope derived cyclic peptides with rationally designed affinity-enhancing substitutions can modulate obligate protein homo-oligomers, which can be used to design peptide inhibitors for other seemingly intractable oligomeric proteins.

Keywords:  cyclic-constrained peptides; loop epitopes; obligate homo-oligomers; protein-protein modulators; structure-based peptide design and optimization

DOI:  https://doi.org/10.3390/molecules28176430
J Org Chem. 2023 Sep 06.

Trifluoromethylthiolation of Tryptophan and Tyrosine Derivatives: A Tool for Enhancing the Local Hydrophobicity of Peptides.

Jure Gregorc, Nathalie Lensen, Grégory Chaume, Jernej Iskra, Thierry Brigaud.

The incorporation of fluorinated groups into peptides significantly affects their biophysical properties. We report herein the synthesis of Fmoc-protected trifluoromethylthiolated tyrosine (CF3S-Tyr) and tryptophan (CF3S-Trp) analogues on a gram scale (77-93% yield) and demonstrate their use as highly hydrophobic fluorinated building blocks for peptide chemistry. The developed methodology was successfully applied to the late-stage regioselective trifluoromethylthiolation of Trp residues in short peptides (66-80% yield) and the synthesis of various CF3S-analogues of biologically active monoamines. To prove the concept, Fmoc-(CF3S)Tyr and -Trp were incorporated into the endomorphin-1 chain (EM-1) and into model tripeptides by solid-phase peptide synthesis. A remarkable enhancement of the local hydrophobicity of the trifluoromethylthiolated peptides was quantified by the chromatographic hydrophobicity index determination method, demonstrating the high potential of CF3S-containing amino acids for the rational design of bioactive peptides.

DOI: https://doi.org/10.1021/acs.joc.3c01373
Methods Mol Biol. 2023 ;2705 269-290

Synthesis and Biochemical Evaluation of Monocarboxylic GRB2 SH2 Domain Inhibitors.

Tao Xiao, Min Zhang, Haitao Ji.

  This protocol discloses the synthesis of monocarboxylic inhibitors with a macrocyclic peptide scaffold to bind with the GRB2 SH2 domain and disrupt the protein-protein interactions (PPIs) between GRB2 and phosphotyrosine-containing proteins.

Keywords:  Diastereomeric selectivity; GRB2; Inhibitors; Macrocyclic peptides; Protein–protein interactions; SH2 domain; Synthesis

DOI:  https://doi.org/10.1007/978-1-0716-3393-9_15
J Chem Inf Model. 2023 Sep 08.

Effect of Spacer Length Modification of the Cationic Side Chain on the Energetics of Antimicrobial Peptide Binding to Membrane-Mimetic Bilayers.

Suvankar Ghosh, Sunanda Chatterjee, Priyadarshi Satpati.

Understanding the mechanism of action of the antimicrobial peptide (AMP) in terms of its structure and energetics is the key to designing new potent and selective AMPs. Recently, we reported a membranolytic 14-residue-long lysine-rich cationic antimicrobial peptide (LL-14: NH3+-LKWLKKLLKWLKKL-CONH2) against Pseudomonas aeruginosa, Klebsiella pneumoniae, and Staphylococcus aureus, which is limited by cytotoxicity and expected to undergo facile protease degradation. Aliphatic side-chain-length modification of the cationic amino-acid residues (Lys and Arg) is a popular strategy for designing protease-resistant AMPs. However, the effect of the peptide side-chain length modifications on the membrane binding affinity and its relation to the atomic structure remain an unsolved problem. We report computer simulations that quantitatively calculated the difference in peptide binding affinity to membrane-mimetic-bilayer models (bacterial: 1-palmitoyl-2-oleoyl-phosphatidylethanolamine (POPE)/1-palmitoyl-2-oleoyl-phosphatidylglycerol (POPG) bilayer and mammalian: 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) bilayer) upon decreasing or increasing the spacer length of the cationic lysine residues of LL-14 (as well as their arginine analogues). We show that the peptide/bilayer interaction energetics varies drastically in response to spacer length modification. The strength of peptide discrimination depends strongly on the nature of the bilayer (bacterial or mammalian mimetic model). An increase in the lysine spacer length by one carbon (i.e., homolysine analogue of LL-14) is weakly/strongly disfavored by the bacterial/mammalian-membrane-mimetic bilayer. Recently, we have demonstrated an excellent correlation between the antimicrobial activity of the membranolytic cationic peptides and their binding affinity to membrane-mimetic-bilayer models. Thus, the homolysine analogue of LL-14 is a promising noncytotoxic AMP with conserved activity. On the other hand, homoarginine analogue (arginine spacer length increment by a single carbon) was preferred by both the bacteria and the mammalian mimetic bilayers and displayed the strongest affinity for the former among the peptides studied in this work. Thus, the promising most potent homoarginine analogue is likely to be cytotoxic. Shortening the Lys/Arg side chain to a three-carbon spacer (Dab/Agb) improves the binding affinity to bacterial and mammalian-membrane-mimetic bilayers. Arginine and arginine-derivative peptides exhibited stronger binding affinity to the bilayers relative to the lysine analogue. The results provide a plausible explanation to the previous experimental observations, viz., superior antimicrobial activity of the arginine peptides relative to Lys peptides and the improvement of antimicrobial activity upon substitution of Lys with Dab in the cationic peptides. The simulations revealed that the small change in the peptide hydrophobicity by Lys/Arg spacer length modification could drastically alter the energetics of peptide/bilayer binding by fine-tuning the electrostatic interactions. The energetics underlying the peptide selectivity by simple membrane-mimetic bilayer models may be beneficial for designing new selective and protease-resistant AMPs.

DOI: https://doi.org/10.1021/acs.jcim.3c01080
Amino Acids. 2023 Sep 05.

PepAnalyzer: predicting peptide properties using its sequence.

Vanshika Rustagi, Shradheya R R Gupta, Monika Bajaj, Archana Singh, Indrakant Kumar Singh.

  Peptides are short linear molecules consisting of amino acids that play an essential role in most biological processes. They can treat diseases by working as a vaccine or antimicrobial agent and serves as a cancer molecule to deliver the drug to the target site for the treatment of cancer. They have the potential to solve the drawbacks of current medications and can be industrially produced in large quantities at low cost. However, poor chemical and physical stability, short circulating plasma half-life, and solubility are some issues that need solutions before they can be used as therapeutics. PepAnalyzer tool is a user-friendly tool that predicts 15 different properties such as binding potential, half-life, transmembrane patterns, test tube stability, charge, isoelectric point, molecular weights, and molar extinction coefficients only using the sequence. The tool is designed using BioPython utility and has even results with standard tools, such as Expasy, EBI, Genecorner, and Geneinfinity. The tool assists students, researchers, and the pharmaceutical sector. The PepAnalyzer tool's online platform is accessible at the link: http://www.iksmbrlabdu.in/peptool .

Keywords:  BLAST; Binding potential; Hydropathy plot; Isoelectric point; Molecular weight; PepAnalyzer; Peptide; Prediction

DOI:  https://doi.org/10.1007/s00726-023-03317-x
Nanomaterials (Basel). 2023 Sep 01. pii: 2476. [Epub ahead of print]13(17):

Peptides Targeting HER2-Positive Breast Cancer Cells and Applications in Tumor Imaging and Delivery of Chemotherapeutics.

Palmira Alessia Cavallaro, Marzia De Santo, Emilia Lucia Belsito, Camilla Longobucco, Manuela Curcio, Catia Morelli, Luigi Pasqua, Antonella Leggio.

  Breast cancer represents the most common cancer type and one of the major leading causes of death in the female worldwide population. Overexpression of HER2, a transmembrane glycoprotein related to the epidermal growth factor receptor, results in a biologically and clinically aggressive breast cancer subtype. It is also the primary driver for tumor detection and progression and, in addition to being an important prognostic factor in women diagnosed with breast cancer, HER2 is a widely known therapeutic target for drug development. The aim of this review is to provide an updated overview of the main approaches for the diagnosis and treatment of HER2-positive breast cancer proposed in the literature over the past decade. We focused on the different targeting strategies involving antibodies and peptides that have been explored with their relative outcomes and current limitations that need to be improved. The review also encompasses a discussion on targeted peptides acting as probes for molecular imaging. By using different types of HER2-targeting strategies, nanotechnology promises to overcome some of the current clinical challenges by developing novel HER2-guided nanosystems suitable as powerful tools in breast cancer imaging, targeting, and therapy.

Keywords:  HER2-positive breast cancer; drug delivery; monoclonal antibodies; nanoparticles; targeting peptides; tumor imaging

DOI:  https://doi.org/10.3390/nano13172476
Bioorg Med Chem. 2023 Aug 26. pii: S0968-0896(23)00309-7. [Epub ahead of print]93 117461

Discovery of a potent, orally available tricyclic derivative as a novel BRD4 inhibitor for melanoma.

Yuhei Horai, Naoki Suda, Shinsuke Uchihashi, Mayako Katakuse, Tomomi Shigeno, Takashige Hirano, Junichi Takahara, Tomoyuki Fujita, Yohei Mukoyama, Yuji Haga.

  The epigenetic regulation of the protein bromodomain-containing protein 4 (BRD4) has emerged as a compelling target for cancer treatment. In this study, we outline the discovery of a novel BRD4 inhibitor for melanoma therapy. Our initial finding was that benzimidazole derivative 1, sourced from our library, was a powerful BRD4 inhibitor. However, it exhibited a poor pharmacokinetic (PK) profile. To address this, we conducted a scaffold-hopping procedure with derivative 1, which resulted in the creation of benzimidazolinone derivative 5. This new derivative displayed an improved PK profile. To further enhance the BRD4 inhibitory activity, we attempted to introduce hydrogen bond acceptors. This indeed improved the activity, but at the cost of decreased membrane permeability. Our search for a potent inhibitor with desirable permeability led to the development of tricyclic 18. This compound demonstrated powerful inhibitory activity and a favorable PK profile. More significantly, tricyclic 18 showed antitumor efficacy in a mouse melanoma xenograft model, suggesting that it holds potential as a therapeutic agent for melanoma treatment.

Keywords:  BRD4 inhibitors; Hydrogen bond acceptor; Melanoma; Permeability; Pharmacokinetics; Scaffold-hopping; Structure-activity relationship

DOI:  https://doi.org/10.1016/j.bmc.2023.117461
Methods Mol Biol. 2023 ;2705 359-369

Peptides as Baits for the Coprecipitation of SH2 Domain-Containing Proteins.

Azin Kiani, Pablo Rios, Maja Köhn.

  Phosphotyrosine (pTyr)-containing amino acid sequences have regulatory effects on proteins that contain pTyr recognition motifs, such as Src Homology 2 (SH2) domains. Using pTyr-containing peptides as a bait for coprecipitation, by immobilization of the synthesized phosphopeptides to beads and incubation with cell lysates, enables to study the binding preference of the SH2 domain for the specific pTyr-sequence obtained from a pTyr-containing protein in a complex biological environment. Using phosphopeptides allows to not only assess the wild-type sequence, but also peptides that can contain modified sequences which carry a nonhydrolyzable pTyr or other modifications varying the binding strength and selectivity, for example, to create strong SH2 domain binders to inhibit their interaction with pTyr-containing proteins. This pulldown experiment can be used as an assay to evaluate the ability of a peptide to bind to the protein of interest in the cell lysate or investigate the selectivity of the peptide. Therefore, immobilizing phosphopeptides and using them as a pulldown tool has a wide range of applications.

Keywords:  Immobilization; Pulldown; Selectivity; Src Homology 2 (SH2) domains; pTyr

DOI:  https://doi.org/10.1007/978-1-0716-3393-9_19