bims-cepepe 2023-10-08 papers

bims-cepepe

Biomed News

on Cell-penetrating peptides

Issue of 2023–10–08
twelve papers selected by
Henry Lamb, Queensland University of Technology

Magainin 2-derived stapled peptides derived with the ability to deliver pDNA, mRNA, and siRNA into cells.
Why do polyarginines adsorb at neutral phospholipid bilayers and polylysines do not? An insight from density functional theory calculations and molecular dynamics simulations.
Inkjet-Based Intracellular Delivery System that Effectively Utilizes Cell-Penetrating Peptides for Cytosolic Introduction of Biomacromolecules through the Cell Membrane.
Deep mutational scanning and machine learning uncover antimicrobial peptide features driving membrane selectivity.
Selective thiazoline peptide cyclisation compatible with mRNA display and efficient synthesis.
Resistance is futile: targeting multidrug-resistant bacteria with de novo Cys-rich cyclic polypeptides.
A multitier virtual screening of antagonists targeting PD-1/PD-L1 interface for the management of triple-negative breast cancer.
Backbone N-methylation of peptides: Advances in synthesis and applications in pharmaceutical drug development.
Designed Endocytosis-Triggering Proteins mediate Targeted Degradation.
Digestion-resistant whey peptides promote antioxidant effect on Caco-2 cells.
Specific peptide conjugation to a therapeutic antibody leads to enhanced therapeutic potency and thermal stability by reduced Fc dynamics.
Construction of a T7 phage random peptide library by combining seamless cloning with in vitro translation.

Chem Sci. 2023 Oct 04. 14(38): 10403-10410

Magainin 2-derived stapled peptides derived with the ability to deliver pDNA, mRNA, and siRNA into cells.

Motoharu Hirano, Hidetomo Yokoo, Chihiro Goto, Makoto Oba, Takashi Misawa, Yosuke Demizu.

We have developed cell-penetrating stapled peptides based on the amphipathic antimicrobial peptide magainin 2 for intracellular delivery of nucleic acids such as pDNA, mRNA, and siRNA. Various types of stapled peptides with a cross-linked structure were synthesised in the hydrophobic region of the amphipathic structure, and their efficacy in intracellular delivery of pDNA was evaluated. The results showed that the stapled peptide st7-5 could deliver pDNA into cells. To improve the deliverability of st7-5, we further designed st7-5_R, in which the Lys residues were replaced by Arg residues. The peptide st7-5_R formed compact and stable complexes with pDNA and was able to efficiently transfer pDNA into the cell. In addition to pDNA, st7-5_R was also able to deliver mRNA and siRNA into the cell. Thus, st7-5_R is a novel peptide that can achieve efficient intracellular delivery of three different nucleic acids.

DOI: https://doi.org/10.1039/d3sc04124g
Phys Chem Chem Phys. 2023 Oct 04.

Why do polyarginines adsorb at neutral phospholipid bilayers and polylysines do not? An insight from density functional theory calculations and molecular dynamics simulations.

Carmelo Tempra, Zlatko Brkljača, Mario Vazdar.

Adsorption of cell-penetrating peptides (CPPs) at cellular membranes is the first and necessary step for their subsequent translocation across cellular membranes into the cytosol. It has been experimentally shown that CPPs rich in arginine (Arg) amino acid penetrate across phospholipid bilayers more effectively than their lysine (Lys) rich counterparts. In this work, we aim to understand the differences in the first translocation step, adsorption of Arg9 and Lys9 peptides at fully hydrated neutral phosphatidylcholine (PC) and phosphatidylethanolamine (PE) lipid bilayers and evaluate in detail the energetics of the process using molecular dynamics (MD) simulations and free energy calculations of adsorption of the single peptide. We show that the adsorption of Arg9 is energetically feasible, with the free energy of adsorption being ∼-5.0 kcal mol-1 at PC and ∼-5.5 kcal mol-1 at PE bilayers. In contrast, adsorption of Lys9 is not observed at PC bilayers, and their adsorption at PE bilayers is very weak, being ∼-0.5 kcal mol-1. We show by energy decomposition and analysis of peptide hydration along the membrane that significantly stronger electrostatic interactions of Arg9 with lipid phosphate groups, together with the greater loss of peptide hydration (and in turn stronger hydrophobic interactions) along the membrane translocation path, are the main driving factors governing the adsorption of Arg-rich peptides at neutral lipid bilayers in contrast to Lys-rich peptides. Finally, we also compare the energetics in lipid/bilayer systems with the density functional theory (DFT) calculations of the corresponding model systems in the continuum water model and reveal the energetic differences in different environments.

DOI: https://doi.org/10.1039/d3cp02411c
ACS Appl Mater Interfaces. 2023 Oct 04.

Inkjet-Based Intracellular Delivery System that Effectively Utilizes Cell-Penetrating Peptides for Cytosolic Introduction of Biomacromolecules through the Cell Membrane.

Mika Omura, Kenta Morimoto, Yurina Araki, Hisaaki Hirose, Yoshimasa Kawaguchi, Yukiya Kitayama, Yuto Goto, Atsushi Harada, Ikuo Fujii, Tomoka Takatani-Nakase, Shiroh Futaki, Ikuhiko Nakase.

  In the drug delivery system, the cytosolic delivery of biofunctional molecules such as enzymes and genes must achieve sophisticated activities in cells, and microinjection and electroporation systems are typically used as experimental techniques. These methods are highly reliable, and they have high intracellular transduction efficacy. However, a high degree of proficiency is necessary, and induced cytotoxicity is considered as a technical problem. In this research, a new intracellular introduction technology was developed through the cell membrane using an inkjet device and cell-penetrating peptides (CPPs). Using the inkjet system, the droplet volume, droplet velocity, and dropping position can be accurately controlled, and minute samples (up to 30 pL/shot) can be carried out by direct administration. In addition, CPPs, which have excellent cell membrane penetration functions, can deliver high-molecular-weight drugs and nanoparticles that are difficult to penetrate through the cell membrane. By using the inkjet system, the CPPs with biofunctional cargo, including peptides, proteins such as antibodies, and exosomes, could be accurately delivered to cells, and efficient cytosolic transduction was confirmed.

Keywords:  Inkjet system; cell-penetrating peptides; cytosolic delivery; macromolecules; membrane penetration

DOI:  https://doi.org/10.1021/acsami.3c01650
Res Sq. 2023 Sep 13. pii: rs.3.rs-3280212. [Epub ahead of print]

Deep mutational scanning and machine learning uncover antimicrobial peptide features driving membrane selectivity.

Bryan Davies, Justin Randall, Luiz Vieira, Claus Wilke.

Antimicrobial peptides commonly act by disrupting bacterial membranes, but also frequently damage mammalian membranes. Deciphering the rules governing membrane selectivity is critical to understanding their function and enabling their therapeutic use. Past attempts to decipher these rules have failed because they cannot interrogate adequate peptide sequence variation. To overcome this problem, we develop deep mutational surface localized antimicrobial display (dmSLAY), which reveals comprehensive positional residue importance and flexibility across an antimicrobial peptide sequence. We apply dmSLAY to Protegrin-1, a potent yet toxic antimicrobial peptide, and identify thousands of sequence variants that positively or negatively influence its antibacterial activity. Further analysis reveals that avoiding large aromatic residues and eliminating disulfide bound cysteine pairs while maintaining membrane bound secondary structure greatly improves Protegrin-1 bacterial specificity. Moreover, dmSLAY datasets enable machine learning to expand our analysis to include over 5.7 million sequence variants and reveal full Protegrin-1 mutational profiles driving either bacterial or mammalian membrane specificity. Our results describe an innovative, high-throughput approach for elucidating antimicrobial peptide sequence-structure-function relationships which can inform synthetic peptide-based drug design.

DOI: https://doi.org/10.21203/rs.3.rs-3280212/v1
Chem Sci. 2023 Oct 04. 14(38): 10561-10569

Selective thiazoline peptide cyclisation compatible with mRNA display and efficient synthesis.

Minglong Liu, Richard Morewood, Ryoji Yoshisada, Mirte N Pascha, Antonius J P Hopstaken, Eliza Tarcoveanu, David A Poole, Cornelis A M de Haan, Christoph Nitsche, Seino A K Jongkees.

Peptide display technologies are a powerful method for discovery of new bioactive sequences, but linear sequences are often very unstable in a biological setting. Macrocyclisation of such peptides is beneficial for target affinity, selectivity, stability, and cell permeability. However, macrocyclisation of a linear hit is unreliable and requires extensive structural knowledge. Genetically encoding macrocyclisation during the discovery process is a better approach, and so there is a need for diverse cyclisation options that can be deployed in the context of peptide display techniques such as mRNA display. In this work we show that meta-cyanopyridylalanine (mCNP) can be ribosomally incorporated into peptides, forming a macrocycle in a spontaneous and selective reaction with an N-terminal cysteine generated from bypassing the initiation codon in translation. This reactive amino acid can also be easily incorporated into peptides during standard Fmoc solid phase peptide synthesis, which can otherwise be a bottleneck in transferring from peptide discovery to peptide testing and application. We demonstrate the potential of this new method by discovery of macrocyclic peptides targeting influenza haemagglutinin, and molecular dynamics simulation indicates the mCNP cross-link stabilises a beta sheet structure in a representative of the most abundant cluster of active hits. Cyclisation by mCNP is also shown to be compatible with thioether macrocyclisation at a second cysteine to form bicycles of different architectures, provided that cysteine placement reinforces selectivity, with this bicyclisation happening spontaneously and in a controlled manner during peptide translation. Our new approach generates macrocycles with a more rigid cross-link and with better control of regiochemistry when additional cysteines are present, opening these up for further exploitation in chemical modification of in vitro translated peptides, and so is a valuable addition to the peptide discovery toolbox.

DOI: https://doi.org/10.1039/d3sc03117a
RSC Chem Biol. 2023 Oct 04. 4(10): 722-735

Resistance is futile: targeting multidrug-resistant bacteria with de novo Cys-rich cyclic polypeptides.

Alvaro Mourenza, Rajasekaran Ganesan, Julio A Camarero.

The search for novel antimicrobial agents to combat microbial pathogens is intensifying in response to rapid drug resistance development to current antibiotic therapeutics. The use of disulfide-rich head-to-tail cyclized polypeptides as molecular frameworks for designing a new type of peptide antibiotics is gaining increasing attention among the scientific community and the pharmaceutical industry. The use of macrocyclic peptides, further constrained by the presence of several disulfide bonds, makes these peptide frameworks remarkably more stable to thermal, biological, and chemical degradation showing better activities when compared to their linear analogs. Many of these novel peptide scaffolds have been shown to have a high tolerance to sequence variability in those residues not involved in disulfide bonds, able to cross biological membranes, and efficiently target complex biomolecular interactions. Hence, these unique properties make the use of these scaffolds ideal for many biotechnological applications, including the design of novel peptide antibiotics. This article provides an overview of the new developments in the use of several disulfide-rich cyclic polypeptides, including cyclotides, θ-defensins, and sunflower trypsin inhibitor peptides, among others, in the development of novel antimicrobial peptides against multidrug-resistant bacteria.

DOI: https://doi.org/10.1039/d3cb00015j
Med Oncol. 2023 Sep 30. 40(11): 312

A multitier virtual screening of antagonists targeting PD-1/PD-L1 interface for the management of triple-negative breast cancer.

HemaNandini Rajendran Krishnamoorthy, Ramanathan Karuppasamy.

  Immunotherapies are promising therapeutic options for the management of triple-negative breast cancer because of its high mutation rate and genomic instability. Of note, the blockade of the immune checkpoint protein PD-1 and its ligand PD-L1 has been proven to be an efficient and potent strategy to combat triple-negative breast cancer. To date, various anti-PD-1/anti-PD-L1 antibodies have been approved. However, the intrinsic constraints of these therapeutic antibodies significantly limit their application, making small molecules a potentially significant option for PD-1/PD-L1 inhibition. In light of this, the current study aims to use a high-throughput virtual screening technique to identify potential repurposed candidates as PD-L1 inhibitors. Thus, the present study explored binding efficiency of 2509 FDA-approved compounds retrieved from the drug bank database against PD-L1 protein. The binding affinity of the compounds was determined using the glide XP docking programme. Furthermore, prime-MM/GBSA, DFT calculations, and RF score were used to precisely re-score the binding free energy of the docked complexes. In addition, the ADME and toxicity profiles for the lead compounds were also examined to address PK/PD characteristics. Altogether, the screening process identified three molecules, namely DB01238, DB06016 and DB01167 as potential therapeutics for the PD-L1 protein. To conclude, a molecular dynamic simulation of 100 ns was run to characterise the stability and inhibitory action of the three lead compounds. The results from the simulation study confirm the robust structural and thermodynamic stability of DB01238 than other investigated molecules. Thus, our findings hypothesize that DB01238 could serve as potential PD-L1 inhibitor in the near future for triple-negative breast cancer patients.

Keywords:  DFT; Drug repurposing; Immune checkpoint inhibitors; Molecular dynamics; PD-L1

DOI:  https://doi.org/10.1007/s12032-023-02183-7
Bioorg Chem. 2023 Sep 27. pii: S0045-2068(23)00553-9. [Epub ahead of print]141 106892

Backbone N-methylation of peptides: Advances in synthesis and applications in pharmaceutical drug development.

Xuefei Li, Ningchao Wang, Yuhang Liu, Weipiao Li, Xinyu Bai, Ping Liu, Chun-Yang He.

  Peptide-based drugs have garnered considerable attention in recent years owing to their increasingly crucial role in the treatment of diverse diseases. However, the limited pharmacokinetic properties of peptides have hindered their full potential. One prominent strategy for enhancing the druggability of peptides is N-methylation, which involves the addition of a methyl group to the nitrogen atom of the peptide backbone. This modification significantly improves the stability, bioavailability, receptor binding affinity and selectivity of peptide drug candidates. In this review, we provide a comprehensive overview of the advancements in synthetic methods for N-methylated peptide synthesis, as well as the associated limitations. Moreover, we explore the versatile effects of N-methylation on various aspects of peptide properties. Furthermore, we emphasize the efforts dedicated to N-methylated peptide pharmaceuticals that have successfully obtained marketing approval.

Keywords:  N-methylation; Peptide synthesis; Pharmaceutical drugs; Pharmacological impact

DOI:  https://doi.org/10.1016/j.bioorg.2023.106892
bioRxiv. 2023 Sep 20. pii: 2023.08.19.553321. [Epub ahead of print]

Designed Endocytosis-Triggering Proteins mediate Targeted Degradation.

Buwei Huang, Mohamad Abedi, Green Ahn, Brian Coventry, Isaac Sappington, Rong Wang, Thomas Schlichthaerle, Jason Z Zhang, Yujia Wang, Inna Goreshnik, Ching Wen Chiu, Adam Chazin-Gray, Sidney Chan, Stacey Gerben, Analisa Murray, Shunzhi Wang, Jason O'Neill, Ronald Yeh, Ayesha Misquith, Anitra Wolf, Luke M Tomasovic, Dan I Piraner, Maria J Duran Gonzalez, Nathaniel R Bennett, Preetham Venkatesh, Danny Satoe, Maggie Ahlrichs, Craig Dobbins, Wei Yang, Xinru Wang, Dionne Vafeados, Rubul Mout, Shirin Shivaei, Longxing Cao, Lauren Carter, Lance Stewart, Jamie B Spangler, Gonçalo J L Bernardes, Kole T Roybal, Per Greisen, Xiaochun Li, Carolyn Bertozzi, David Baker.

Endocytosis and lysosomal trafficking of cell surface receptors can be triggered by interaction with endogenous ligands. Therapeutic approaches such as LYTAC 1,2 and KineTAC 3 , have taken advantage of this to target specific proteins for degradation by fusing modified native ligands to target binding proteins. While powerful, these approaches can be limited by possible competition with the endogenous ligand(s), the requirement in some cases for chemical modification that limits genetic encodability and can complicate manufacturing, and more generally, there may not be natural ligands which stimulate endocytosis through a given receptor. Here we describe general protein design approaches for designing endocytosis triggering binding proteins (EndoTags) that overcome these challenges. We present EndoTags for the IGF-2R, ASGPR, Sortillin, and Transferrin receptors, and show that fusing these tags to proteins which bind to soluble or transmembrane protein leads to lysosomal trafficking and target degradation; as these receptors have different tissue distributions, the different EndoTags could enable targeting of degradation to different tissues. The modularity and genetic encodability of EndoTags enables AND gate control for higher specificity targeted degradation, and the localized secretion of degraders from engineered cells. The tunability and modularity of our genetically encodable EndoTags should contribute to deciphering the relationship between receptor engagement and cellular trafficking, and they have considerable therapeutic potential as targeted degradation inducers, signaling activators for endocytosis-dependent pathways, and cellular uptake inducers for targeted antibody drug and RNA conjugates.

DOI: https://doi.org/10.1101/2023.08.19.553321
Food Res Int. 2023 Nov;pii: S0963-9969(23)00836-0. [Epub ahead of print]173(Pt 1): 113291

Digestion-resistant whey peptides promote antioxidant effect on Caco-2 cells.

Juliana Santos de Espindola, Milena Ferreira Taccóla, Vera Sônia Nunes da Silva, Lucilene Delazari Dos Santos, Bruno Cesar Rossini, Bruna Cavecci Mendonça, Maria Teresa Bertoldo Pacheco, Fabiana Galland.

  Enteric endothelial cells are the first structure to come in contact with digested food and may suffer oxidative damage by innumerous exogenous factors. Although peptides derived from whey digestion have presented antioxidant potential, little is known regarding antioxidant pathways activation in Caco-2 cell line model. Hence, we evaluated the ability to form whey peptides resistant to simulated gastrointestinal digestive processes, with potential antioxidant activity on gastrointestinal cells and associated with sequence structure and activity. Using the INFOGEST method of simulated static digestion, we achieved 35.2% proteolysis, with formation of peptides of low molecular mass (<600 Da) evaluated by FPLC. The digestion-resistant peptides showed a high proportion of hydrophobic and acidic amino acids, but with average surface hydrophobicity. We identified 24 peptide sequences, mainly originated from β-lactoglobulin, that exhibit various bioactivities. Structurally, the sequenced peptides predominantly contained the amino acids lysine and valine in the N-terminal region, and tyrosine in the C-terminal region, which are known to exhibit antioxidant properties. The antioxidant activity of the peptide digests was on average twice as potent as that of the protein isolates for the same concentration, as evaluated by ABTS, DPPH and ORAC. Evaluation of biological activity in Caco-2 intestinal cells, stimulated with hydrogen peroxide, showed that they attenuated the production of reactive oxygen species and prevented GSH reduction and SOD activity increase. Caco-2 cells were not responsive to nitric oxide secretion. This study suggests that whey peptides formed during gastric digestion exhibit biological antioxidant activity, without the need for previously hydrolysis with exogenous enzymes for supplement application. The study's primary contribution was demonstrating the antioxidant activity of whey peptides in maintaining the gastrointestinal epithelial cells, potentially preventing oxidative stress that affects the digestive system.

Keywords:  Antioxidant pathways; Antioxidant peptides; Caco-2 cells; INFOGEST; Whey hydrolysate

DOI:  https://doi.org/10.1016/j.foodres.2023.113291
Sci Rep. 2023 10 02. 13(1): 16561

Specific peptide conjugation to a therapeutic antibody leads to enhanced therapeutic potency and thermal stability by reduced Fc dynamics.

Masato Kiyoshi, Makoto Nakakido, Abdur Rafique, Minoru Tada, Michihiko Aoyama, Yosuke Terao, Satoru Nagatoishi, Hiroko Shibata, Teruhiko Ide, Kouhei Tsumoto, Yuji Ito, Akiko Ishii-Watabe.

Antibody-drug conjugates are powerful tools for combatting a wide array of cancers. Drug conjugation to a therapeutic antibody often alters molecular characteristics, such as hydrophobicity and effector function, resulting in quality deterioration. To develop a drug conjugation methodology that maintains the molecular characteristics of the antibody, we engineered a specific peptide for conjugation to the Fc region. We used trastuzumab and the chelator (DOTA) as model antibody and payload, respectively. Interestingly, peptide/DOTA-conjugated trastuzumab exhibited enhanced antibody-dependent cellular cytotoxicity (ADCC) and increased thermal stability. Detailed structural and thermodynamic analysis clarified that the conjugated peptide blocks the Fc dynamics like a "wedge." We revealed that (1) decreased molecular entropy results in enhanced ADCC, and (2) blockade of Fc denaturation results in increased thermal stability. Thus, we believe that our methodology is superior not only for drug conjugation but also as for reinforcing therapeutic antibodies to enhance ADCC and thermal stability.

DOI: https://doi.org/10.1038/s41598-023-43431-0
J Biochem. 2023 Oct 05. pii: mvad077. [Epub ahead of print]

Construction of a T7 phage random peptide library by combining seamless cloning with in vitro translation.

Katsuaki Higashi, Sakiho Oda, Mai Fujii, Fumiya Nishida, Hayato Matsumoto, Jyoji Morise, Shogo Oka, Motohiro Nonaka.

  T7 phage libraries displaying random peptides are powerful tools for screening peptide sequences that bind to various target molecules. The T7 phage system has the advantage of less biased peptide distribution compared to the M13 phage system. However, the construction of T7 phage DNA is challenging due to its long 36 kb linear DNA. Furthermore, the diversity of the libraries depends strongly on the efficiency of commercially available packaging extracts. To address these issues, we examined the combination of seamless cloning with cell-free translation systems. Seamless cloning technologies have been widely used to construct short circular plasmid DNA, and several recent studies showed that cell-free translation can achieve more diverse phage packaging. In this study, we combined these techniques to construct four libraries (CX7C, CX9C, CX11C, and CX13C) with different random regions lengths. The libraries thus obtained all showed diversity > 109 plaque forming units (pfu). Evaluating our libraries with an anti-FLAG monoclonal antibody yielded the correct epitope sequence. The results indicate that our libraries are useful for screening peptide epitopes against antibodies. These findings suggest that our system can efficiently construct T7 phage libraries with greater diversity than previous systems.

Keywords:   In vitro translation; Diversity; Peptide screening; Phage library; Seamless cloning

DOI:  https://doi.org/10.1093/jb/mvad077