bims-cepepe 2024-12-01 papers

bims-cepepe

Biomed News

on Cell-penetrating peptides

Issue of 2024–12–01
thirteen papers selected by
Henry Lamb, Queensland University of Technology

Accurate de novo design of high-affinity protein binding macrocycles using deep learning.
Advancements in Loop Cyclization Approaches for Enhanced Peptide Therapeutics for Targeting Protein-Protein Interactions.
Endosomolytic peptides enable the cellular delivery of peptide nucleic acids.
Structural Insights into Helix-Loop-Helix Peptides for "Ligand-Targeting" Intracellular Drug Delivery via VEGF Receptor-Mediated Endocytosis.
Recent Advances in Peptide Drug Discovery: Novel Strategies and Targeted Protein Degradation.
Structure of a Cyclic Peptide as an Inhibitor of Mycobacterium tuberculosis Transcription: NMR and Molecular Dynamics Simulations.
Enhancing Tumor Targeted Therapy: The Role of iRGD Peptide in Advanced Drug Delivery Systems.
Design and functional studies of xylene-based cyclic mimetics of SOCS1 protein.
Designed Cell-Penetrating Peptide Constructs for Inhibition of Pathogenic Protein Self-Assembly.
In vitro selection of dye-fluorescence-enhancing peptide aptamer by cDNA display.
Elucidating the conformational behavior and membrane-destabilizing capability of the antimicrobial peptide ecPis-4s.
High throughput application of the NanoBiT Biochemical Assay for the discovery of selective inhibitors of the interaction of PI3K-p110α with KRAS.
Interpreting the function of cell penetrating peptide (RGD) in drug transport to the cell membrane: a computational approach.

bioRxiv. 2024 Nov 18. pii: 2024.11.18.622547. [Epub ahead of print]

Accurate de novo design of high-affinity protein binding macrocycles using deep learning.

Stephen A Rettie, David Juergens, Victor Adebomi, Yensi Flores Bueso, Qinqin Zhao, Alexandria N Leveille, Andi Liu, Asim K Bera, Joana A Wilms, Alina Üffing, Alex Kang, Evans Brackenbrough, Mila Lamb, Stacey R Gerben, Analisa Murray, Paul M Levine, Maika Schneider, Vibha Vasireddy, Sergey Ovchinnikov, Oliver H Weiergräber, Dieter Willbold, Joshua A Kritzer, Joseph D Mougous, David Baker, Frank DiMaio, Gaurav Bhardwaj.

The development of macrocyclic binders to therapeutic proteins typically relies on large-scale screening methods that are resource-intensive and provide little control over binding mode. Despite considerable progress in physics-based methods for peptide design and deep-learning methods for protein design, there are currently no robust approaches for de novo design of protein-binding macrocycles. Here, we introduce RFpeptides, a denoising diffusion-based pipeline for designing macrocyclic peptide binders against protein targets of interest. We test 20 or fewer designed macrocycles against each of four diverse proteins and obtain medium to high-affinity binders against all selected targets. Designs against MCL1 and MDM2 demonstrate K D between 1-10 μM, and the best anti-GABARAP macrocycle binds with a K D of 6 nM and a sub-nanomolar IC 50 in vitro . For one of the targets, RbtA, we obtain a high-affinity binder with K D < 10 nM despite starting from the target sequence alone due to the lack of an experimentally determined target structure. X-ray structures determined for macrocycle-bound MCL1, GABARAP, and RbtA complexes match very closely with the computational design models, with three out of the four structures demonstrating Ca RMSD of less than 1.5 Å to the design models. In contrast to library screening approaches for which determining binding mode can be a major bottleneck, the binding modes of RFpeptides-generated macrocycles are known by design, which should greatly facilitate downstream optimization. RFpeptides thus provides a powerful framework for rapid and custom design of macrocyclic peptides for diagnostic and therapeutic applications.

DOI: https://doi.org/10.1101/2024.11.18.622547
J Org Chem. 2024 Nov 29.

Advancements in Loop Cyclization Approaches for Enhanced Peptide Therapeutics for Targeting Protein-Protein Interactions.

Lucia Lombardi, Luke A Granger, Robin J Shattock, Daryl R Williams.

Protein-protein interactions (PPIs) are pivotal in regulating cellular functions and life processes, making them promising therapeutic targets in modern medicine. Despite their potential, developing PPI inhibitors poses significant challenges due to their large and shallow interfaces that complicate ligand binding. This study focuses on mimicking peptide loops as a strategy for PPI inhibition, utilizing synthetic peptide loops for replicating critical binding regions. This work explores turn-inducing elements and highlights the importance of proline in promoting favorable conformations for lactamization, yielding high-purity cyclic peptides. Notably, our one-pot method offers enhanced versatility and represents a robust strategy for efficient and selective macrolactamization, expanding the scope of peptide synthesis methodologies. This approach, validated through the synthesis of AAV capsid-derived loops, offers a robust platform for developing peptide-based therapeutics and highlights the potential of peptide macrocycles in overcoming PPI drug discovery challenges and advancing the development of new therapeutics.

DOI: https://doi.org/10.1021/acs.joc.4c02178
Chem Commun (Camb). 2024 Nov 27.

Endosomolytic peptides enable the cellular delivery of peptide nucleic acids.

JoLynn B Giancola, Ronald T Raines.

Precision genetic medicine enlists antisense oligonucleotides (ASOs) to bind to nucleic acid targets important for human disease. Peptide nucleic acids (PNAs) have many desirable attributes as ASOs but lack cellular permeability. Here, we use an assay based on the corrective splicing of an mRNA to assess the ability of synthetic peptides to deliver a functional PNA into a human cell. We find that the endosomolytic peptides L17E and L17ER4 are highly efficacious delivery vehicles. Co-treatment of a PNA with low micromolar L17E or L17ER4 enables robust corrective splicing in nearly all treated cells. Peptide-PNA conjugates are even more effective. These results enhance the utility of PNAs as research tools and potential therapeutic agents.

DOI: https://doi.org/10.1039/d4cc05214e
Biochem Biophys Res Commun. 2024 Nov 12. pii: S0006-291X(24)01516-X. [Epub ahead of print]741 150980

Structural Insights into Helix-Loop-Helix Peptides for "Ligand-Targeting" Intracellular Drug Delivery via VEGF Receptor-Mediated Endocytosis.

Masataka Michigami, Ryoichi Kira, Masayuki Kamo, Takatsugu Hirokawa, Takayoshi Kinoshita, Koji Inaka, Ikuhiko Nakase, Ikuo Fujii.

  As a new alternative to antibody-drug conjugates, we developed "ligand-targeting" peptide-drug conjugates (PDCs), in which conformationally constrained helix-loop-helix (HLH) peptide M49 targeting human vascular endothelial growth factor-A (VEGF) was used as a drug carrier. The biochemical study showed that HLH peptide M49 made a complex with VEGF in the extracellular environment, and then the M49/VEGF complex interacts with the receptor on the cell surface to induce cellular internalization via the endocytic pathway. Here, we present an X-ray crystal structure of the M49/VEGF complex at 1.5 Å resolution using a protein crystal grown in microgravity. The structure illustrated the "ligand-targeting" cellular uptake mechanism for intracellular drug delivery and the molecular basis on the peptide-VEGF binding mode with tight binding and high target specificity. In addition, mutational studies and thermodynamic analysis provided information on the driving forces of the complex formation. This work would contribute to the design of mid-size molecular-targeting peptides as well as HLH peptides, advancing the research in drug discovery and chemical biology.

Keywords:  Helix–loop–helix; Peptide-drug conjugate; VEGF; X-ray crystal structure

DOI:  https://doi.org/10.1016/j.bbrc.2024.150980
Pharmaceutics. 2024 Nov 20. pii: 1486. [Epub ahead of print]16(11):

Recent Advances in Peptide Drug Discovery: Novel Strategies and Targeted Protein Degradation.

Katarina Vrbnjak, Raj Nayan Sewduth.

  Recent technological advancements, including computer-assisted drug discovery, gene-editing techniques, and high-throughput screening approaches, have greatly expanded the palette of methods for the discovery of peptides available to researchers. These emerging strategies, driven by recent advances in bioinformatics and multi-omics, have significantly improved the efficiency of peptide drug discovery when compared with traditional in vitro and in vivo methods, cutting costs and improving their reliability. An added benefit of peptide-based drugs is the ability to precisely target protein-protein interactions, which are normally a particularly challenging aspect of drug discovery. Another recent breakthrough in this field is targeted protein degradation through proteolysis-targeting chimeras. These revolutionary compounds represent a noteworthy advancement over traditional small-molecule inhibitors due to their unique mechanism of action, which allows for the degradation of specific proteins with unprecedented specificity. The inclusion of a peptide as a protein-of-interest-targeting moiety allows for improved versatility and the possibility of targeting otherwise undruggable proteins. In this review, we discuss various novel wet-lab and computational multi-omic methods for peptide drug discovery, provide an overview of therapeutic agents discovered through these cutting-edge techniques, and discuss the potential for the therapeutic delivery of peptide-based drugs.

Keywords:  PROTACs; micropeptides; multi-omics; peptide drug delivery; peptide drug design; peptide drugs; targeted protein degradation

DOI:  https://doi.org/10.3390/pharmaceutics16111486
Pharmaceuticals (Basel). 2024 Nov 18. pii: 1545. [Epub ahead of print]17(11):

Structure of a Cyclic Peptide as an Inhibitor of Mycobacterium tuberculosis Transcription: NMR and Molecular Dynamics Simulations.

Filia Stephanie, Usman Sumo Friend Tambunan, Krzysztof Kuczera, Teruna J Siahaan.

   BACKGROUND AND OBJECTIVES: A novel antitubercular cyclic peptide, Cyclo(1,6)-Ac-CLYHFC-NH2, was designed to bind at the rifampicin (RIF) binding site on the RNA polymerase (RNAP) of Mycobacterium tuberculosis (MTB). This peptide inhibits RNA elongation in the MTB transcription initiation assay in the nanomolar range, which can halt the MTB transcription initiation complex, similar to RIF. Therefore, determining the solution conformation of this peptide is useful in improving the peptide's binding affinity to the RNAP.
METHODS: Here, the solution structure of Cyclo(1,6)-Ac-CLYHFC-NH2 was determined by two-dimensional (2D) NMR experiments and NMR-restrained molecular dynamic (MD) simulations.
RESULTS: All protons of Cyclo(1,6)-Ac-CLYHFC-NH2 were assigned using TOCSY and NOE NMR spectroscopy. The NOE cross-peak intensities were used to calculate interproton distances within the peptide. The JNH-HCα coupling constants were used to determine the possible Phi angles within the peptide. The interproton distances and calculated Phi angles from NMR were used in NMR-restrained MD simulations. The NOE spectra showed NH-to-NH cross-peaks at Leu2-to-Tyr3 and Tyr3-to-His4, indicating a βI-turn formation at the Cys1-Leu2-Tyr3-His4 sequence.
CONCLUSIONS: The NMR-restrained MD simulations showed several low-energy conformations that were congruent with the NMR data. Finally, the conformation of this peptide will be used to design derivatives that can better inhibit RNAP activity.

Keywords:  Mycobacterium tuberculosis; cyclic peptide conformation; mRNA polymerase inhibitor; molecular dynamics simulation; nuclear magnetic resonance

DOI:  https://doi.org/10.3390/ph17111545
Cancers (Basel). 2024 Nov 08. pii: 3768. [Epub ahead of print]16(22):

Enhancing Tumor Targeted Therapy: The Role of iRGD Peptide in Advanced Drug Delivery Systems.

Dragana Nikitovic, Ekaterina Kukovyakina, Aikaterini Berdiaki, Alexandros Tzanakakis, Anna Luss, Elizaveta Vlaskina, Anne Yagolovich, Aristides Tsatsakis, Andrey Kuskov.

  Chemotherapy remains the primary therapeutic approach in treating cancer. The tumor microenvironment (TME) is the complex network surrounding tumor cells, comprising various cell types, such as immune cells, fibroblasts, and endothelial cells, as well as ECM components, blood vessels, and signaling molecules. The often stiff and dense network of the TME interacts dynamically with tumor cells, influencing cancer growth, immune response, metastasis, and resistance to therapy. The effectiveness of the treatment of solid tumors is frequently reduced due to the poor penetration of the drug, which leads to attaining concentrations below the therapeutic levels at the site. Cell-penetrating peptides (CPPs) present a promising approach that improves the internalization of therapeutic agents. CPPs, which are short amino acid sequences, exhibit a high ability to pass cell membranes, enabling them to deliver drugs efficiently with minimal toxicity. Specifically, the iRGD peptide, a member of CPPs, is notable for its capacity to deeply penetrate tumor tissues by binding simultaneously integrins ανβ3/ανβ5 and neuropilin receptors. Indeed, ανβ3/ανβ5 integrins are characteristically expressed by tumor cells, which allows the iRGD peptide to home onto tumor cells. Notably, the respective dual-receptor targeting mechanism considerably increases the permeability of blood vessels in tumors, enabling an efficient delivery of co-administered drugs or nanoparticles into the tumor mass. Therefore, the iRGD peptide facilitates deeper drug penetration and improves the efficacy of co-administered therapies. Distinctively, we will focus on the iRGD mechanism of action, drug delivery systems and their application, and deliberate future perspectives in developing iRGD-conjugated therapeutics. In summary, this review discusses the potential of iRGD in overcoming barriers to drug delivery in cancer to maximize treatment efficiency while minimizing side effects.

Keywords:  cancer therapy; cell-penetrating peptides (CPPs); iRGD; nanocarriers; tumor microenvironment

DOI:  https://doi.org/10.3390/cancers16223768
Eur J Med Chem. 2024 Nov 25. pii: S0223-5234(24)00989-9. [Epub ahead of print]282 117107

Design and functional studies of xylene-based cyclic mimetics of SOCS1 protein.

Alessia Cugudda, Sara La Manna, Marilisa Leone, Marian Vincenzi, Daniela Marasco.

  Peptidomimetics of Suppressors of cytokine signaling 1 (SOCS1) protein demonstrated valid therapeutic potentials as anti-inflammatory agents. Indeed, SOCS1 has a small kinase inhibitory region (KIR) primarily involved in the inhibition of the JAnus Kinase/Signal Transducer and Activator of Transcription (JAK/STAT) pathway Herein, on the basis of previous investigations on a potent mimetic of KIR-SOCS1, named PS5, we designed and evaluated the SAR (Structure Activity Relationship) features of two xylene-based macrocycles analogues of PS5. These novel compounds bear thiol-xylene linkages with mono- and bi-cyclic scaffolds: they were in vitro functionally investigated toward JAK2 catalytic domain, as ligands with microscale thermophoresis (MST) and as inhibitors through LC-MS analyses. To evaluate structural properties Circular Dichroism (CD) and Nuclear Magnetic Resonance (NMR) spectroscopies were employed along with serum stability assays. Results indicated that a monocycle scaffold is well-tolerated by PS5 sequence enhancing the affinity toward the kinase with a KD in the low micromolar range and providing consistent inhibitory effects of the catalytic activity, which were evaluated for the first time in the case of SOCS1 mimetics. Conformationally, the presence of xylene scaffold affects the flexibility of the compounds and their stabilities to proteases degradation. This study contributes to the understanding of the factors necessary for accurately mimicking the inhibitory mechanism of SOCS1 protein towards JAK2 and to the translation of proteomimetics into drugs.

Keywords:  Cyclic peptides; Cytokine signaling; JAK-STAT; Mimetic peptides; SOCS1

DOI:  https://doi.org/10.1016/j.ejmech.2024.117107
Pharmaceutics. 2024 Nov 12. pii: 1443. [Epub ahead of print]16(11):

Designed Cell-Penetrating Peptide Constructs for Inhibition of Pathogenic Protein Self-Assembly.

Mona Kalmouni, Yujeong Oh, Wael Alata, Mazin Magzoub.

  Peptides possess a number of pharmacologically desirable properties, including greater chemical diversity than other biomolecule classes and the ability to selectively bind to specific targets with high potency, as well as biocompatibility, biodegradability, and ease and low cost of production. Consequently, there has been considerable interest in developing peptide-based therapeutics, including amyloid inhibitors. However, a major hindrance to the successful therapeutic application of peptides is their poor delivery to target tissues, cells or subcellular organelles. To overcome these issues, recent efforts have focused on engineering cell-penetrating peptide (CPP) antagonists of amyloidogenesis, which combine the attractive intrinsic properties of peptides with potent therapeutic effects (i.e., inhibition of amyloid formation and the associated cytotoxicity) and highly efficient delivery (to target tissue, cells, and organelles). This review highlights some promising CPP constructs designed to target amyloid aggregation associated with a diverse range of disorders, including Alzheimer's disease, transmissible spongiform encephalopathies (or prion diseases), Parkinson's disease, and cancer.

Keywords:  Alzheimer’s disease; Parkinson’s disease; alpha-synuclein; amyloid-beta peptide; cancer; mutant p53; neurodegeneration; prion; protein aggregation; tau

DOI:  https://doi.org/10.3390/pharmaceutics16111443
Anal Biochem. 2024 Nov 22. pii: S0003-2697(24)00266-5. [Epub ahead of print] 115722

In vitro selection of dye-fluorescence-enhancing peptide aptamer by cDNA display.

Takashi Kubo, Tomoyuki Koike, Tomoki Ouchi, Nayab Khaliq, Eita Sasaki, Kouichi Kuroda, Mitsuyoshi Ueda, Kenjiro Hanaoka, Naoto Nemoto.

  Although Green Fluorescent Protein (GFP) is useful and most widely used, steric hindrance due to its size and the time required for chromophore formation are complications. However, it is difficult to form chromophores with peptides to reduce the molecular weight. Therefore, we focused on peptides that can become fluorescent by binding to dyes. In this study, a novel dye-fluorescence-enhancing peptide aptamer was selected by the cDNA display method, which was confirmed by the yeast surface display method. This peptide aptamer binds to the non-fluorescent dye QSY®9 and enhances its fluorescence by preventing rotation of its benzene sulfone group. The method described in this paper should enable the development of new cell imaging methods using non-fluorescent dyes and peptides.

Keywords:  cDNA display; cell imaging; non-fluorescent dye; peptide aptamer

DOI:  https://doi.org/10.1016/j.ab.2024.115722
Biophys Chem. 2024 Nov 14. pii: S0301-4622(24)00182-0. [Epub ahead of print]317 107353

Elucidating the conformational behavior and membrane-destabilizing capability of the antimicrobial peptide ecPis-4s.

K R de Souza, L O Nunes, E S Salnikov, H M Mundim, V H O Munhoz, L M Lião, C Aisenbrey, J M Resende, B Bechinger, R M Verly.

  Here we present studies of the structure and membrane interactions of ecPis-4 s, a new antimicrobial peptide from the piscidin family, which shows a wide-range of potential biotechnological applications. In order to understand the mode of action ecPis-4 s, the peptide was chemically synthesized and structural investigations in the presence of anionic POPC:POPG (3:1, mol:mol) membrane and SDS micelles were performed. CD spectroscopy demonstrated that ecPis-4 s has a high content of helical structure in both membrane mimetic media, which is in line with solution NMR spectroscopy that revealed an amphipathic helical conformation throughout the entire peptide chain. Solid-state NMR experiments of ecPis-4 s selectively labeled with 15N/2H and reconstituted into uniaxially oriented POPC:POPG membranes revealed an ideal partition of hydrophilic and hydrophobic residues within the bilayer interface. The peptide aligns in parallel to the membrane surface, a topology stabilized by aromatic side-chain interactions of the Phe-1, Phe-2 and Trp-9 with the phospholipids. 2H NMR experiments using deuterated lipids revealed that anionic lipid accumulates in the vicinity of the cationic peptide upon peptide-membrane binding.

Keywords:  Antimicrobial peptides; Conformational analysis of peptides; Membrane active peptides; Peptide topology; Peptide-membrane interaction; Piscidins peptides

DOI:  https://doi.org/10.1016/j.bpc.2024.107353
SLAS Discov. 2024 Nov 27. pii: S2472-5552(24)00059-5. [Epub ahead of print] 100197

High throughput application of the NanoBiT Biochemical Assay for the discovery of selective inhibitors of the interaction of PI3K-p110α with KRAS.

Mohamed Ismail, Gareth Davies, Graham Sproat, Tiziana Monteverde, Jonathan Tart, Marta Acebrón-García-de-Eulate, Andrea Gohlke, David Hancock, Santosh Adhikari, Sandra Stefanovic-Barrett, David M Smith, Vikki Flemington, Emma S Gleave-Hanford, Geoffrey A Holdgate, Jason G Kettle, Julian Downward.

  The NanoBiT Biochemical Assay (NBBA) was designed as a biochemical format of the NanoBiT cellular assay, aiming to screen weak protein-protein interactions (PPIs) in mammalian cell lysates. Here we present a High Throughput Screening (HTS) application of the NBBA to screen small molecule and fragment libraries to identify compounds that block the interaction of KRAS-G12D with phosphatidylinositol 3-kinase (PI3K) p110α. This interaction promotes PI3K activity, resulting in the promotion of cell growth, proliferation and survival, and is required for tumour initiation and growth in mouse lung cancer models, whilst having little effect on the health of normal adult mice, establishing the significance of the p110α/KRAS interaction as an oncology drug target. Despite the weak binding affinity of the p110α/KRAS interaction (KD = 3 μM), the NBBA proved to be robust and displayed excellent Z'-factor statistics during the HTS primary screening of 726,000 compounds, which led to the identification of 8,000 active compounds. A concentration response screen comparing KRAS/p110α with two closely related PI3K isoforms, p110δ and p110γ, identified selective p110α-specific compounds and enabled derivation of an IC50 for these hits. We identified around 30 compounds showing greater than 20-fold selectivity towards p110α versus p110δ and p110γ with IC50 < 2 μM. By using Differential Scanning Fluorimetry (DSF) we confirmed several compounds that bind directly to purified p110α. The most potent hits will be followed up by co-crystallization with p110α to aid further elucidation of the nature of the interaction and extended optimisation of these compounds.

Keywords:  Drug Discovery; High throughput screening; KRAS; NanoBiT assay; PI 3-kinase; assay development

DOI:  https://doi.org/10.1016/j.slasd.2024.100197
Sci Rep. 2024 Nov 29. 14(1): 29668

Interpreting the function of cell penetrating peptide (RGD) in drug transport to the cell membrane: a computational approach.

Fatemeh Talaei, Farzaneh Farzad.

  Carbon nanotubes (CNT) have unique properties that make them an excellent option for use as drug carriers. However, to make them safe for the human body, their walls are typically coated with a layer of peptide, which also helps to neutralize their toxicity. Additionally, a specific peptide sequence can be used to deliver therapeutic agents exclusively to cancer cells. In recent years, considerable progress has been made in the development of drug delivery systems (DDS) for drug delivery by computer-assisted. The present study inquires about the loading of ketoprofen (Ket) and naproxen (Nap) conjugated with RGD peptide sequence on CNT and its interaction with the double-layer membrane using the molecular dynamics (MD) simulation method. The obtained results show that the investigated complexes often interact through van der Waals and π-π interactions. Energy values for ketoprofen and naproxen with CNT were evaluated - 270.63 and - 195.8 kJ/mol, respectively. The results of the physical adsorption of the complexes on the membrane surface show that the CNT-KRG and CNT-NRG complexes spontaneously diffuse into the biological membrane. In addition, the study of the interaction energy values of these two complexes with the membrane shows that the van der Waals energy plays a significant role in the stability of the systems. On the other hand, the study of the interaction between the drug-CNT complex and the membrane surface shows that the drug can easily penetrate the membrane in the presence of the peptide sequence and the carrier.

Keywords:  Carbon nanotubes; Drug delivery systems; MD simulation; Metadynamic; Naproxen

DOI:  https://doi.org/10.1038/s41598-024-80060-7