bims-cepepe 2024-09-29 papers

bims-cepepe

Biomed News

on Cell-penetrating peptides

Issue of 2024–09–29
thirteen papers selected by
Henry Lamb, Queensland University of Technology

Next Generation SICLOPPS Screening for the Identification of Inhibitors of the HIF-1α/HIF-1β Protein-Protein Interaction.
Peptide inhibitors targeting Ras and Ras-associated protein-protein interactions.
Stapled Peptides: An Innovative and Ultimate Future Drug Offering a Highly Powerful and Potent Therapeutic Alternative.
Stapled Peptides as Inhibitors of mRNA Deadenylation.
Structure Derivatization of IgG-Binding Peptides and Analysis of Their Secondary Structure by Circular Dichroism Spectroscopy.
Amphiphilic shuttle peptide delivers base editor ribonucleoprotein to correct the CFTR R553X mutation in well-differentiated airway epithelial cells.
A yeast two-hybrid system to obtain triple-helical ligands from combinatorial random peptide libraries.
Blood-brain barrier and blood-brain tumor barrier penetrating peptide-drug conjugate as targeted therapy for the treatment of lung cancer brain metastasis.
PSMA and Sigma-1 receptor dual-targeted peptide mediates superior radionuclide imaging and therapy of prostate cancer.
Developing New Peptides and Peptide-Drug Conjugates for Targeting the FGFR2 Receptor-Expressing Tumor Cells and 3D Spheroids.
Multistate and functional protein design using RoseTTAFold sequence space diffusion.
Application of Single Cell Type-Derived Spheroids Generated by Using a Hanging Drop Culture Technique in Various In Vitro Disease Models: A Narrow Review.
Development of DuoMYC: a synthetic cell penetrant miniprotein that efficiently inhibits the oncogenic transcription factor MYC.

ACS Chem Biol. 2024 Sep 23.

Next Generation SICLOPPS Screening for the Identification of Inhibitors of the HIF-1α/HIF-1β Protein-Protein Interaction.

Alexander McDermott, Leonie M Windeln, Jacob S D Valentine, Leonardo Baldassarre, Andrew D Foster, Ali Tavassoli.

Split-intein circular ligation of proteins and peptides (SICLOPPS) is a method for generating intracellular libraries of cyclic peptides that has yielded several first-in-class inhibitors. Here, we detail a revised high-content, high-throughput SICLOPPS screening protocol that utilizes next-generation sequencing, biopanning, and computational tools to identify hits against a given protein-protein interaction. We used this platform for the identification of inhibitors of the HIF-1α/HIF-1β protein-protein interaction. The revised platform resulted in a significantly higher positive hit rate than that previously reported for SICLOPPS screens, and the identified cyclic peptides were more active in vitro and in cells than our previously reported inhibitors. The platform detailed here may be used for the identification of inhibitors of a wide range of other targets.

DOI: https://doi.org/10.1021/acschembio.4c00494
Eur J Med Chem. 2024 Sep 19. pii: S0223-5234(24)00759-1. [Epub ahead of print]279 116878

Peptide inhibitors targeting Ras and Ras-associated protein-protein interactions.

Dan Han, Anpeng Li, Lie Zhu, Chunlin Zhuang, Qingjie Zhao, Yan Zou.

  Peptides represent attractive molecules for targeting protein-protein interactions, and peptide drug development has made great progress during the last decades. Ras protein, the most promising target in cancer therapy, is one of the major growth drivers in various cancers. Although many small molecule inhibitors have been reported to effectively target Ras protein and some inhibitors (such as MRTX849 and AMG 510) have been translated into clinical application, just a few peptide inhibitors have been reported. Here we summarize different types of peptide inhibitors, including monocyclic peptides, bicyclic peptides, stapled peptides, and proteomimetic inhibitors, developed in recent years; emphasize the limits and achievements; and discuss the outlook and challenges associated with future research in peptide inhibitors. This review aims to provide a reference for the discovery of Ras peptide inhibitors.

Keywords:  Anticancer; Inhibitors; Peptides; Protein–protein interaction; RAS

DOI:  https://doi.org/10.1016/j.ejmech.2024.116878
Biomimetics (Basel). 2024 Sep 05. pii: 537. [Epub ahead of print]9(9):

Stapled Peptides: An Innovative and Ultimate Future Drug Offering a Highly Powerful and Potent Therapeutic Alternative.

Do-Hee Kim, Sung-Min Kang.

  Peptide-based therapeutics have traditionally faced challenges, including instability in the bloodstream and limited cell membrane permeability. However, recent advancements in α-helix stapled peptide modification techniques have rekindled interest in their efficacy. Notably, these developments ensure a highly effective method for improving peptide stability and enhancing cell membrane penetration. Particularly in the realm of antimicrobial peptides (AMPs), the application of stapled peptide techniques has significantly increased peptide stability and has been successfully applied to many peptides. Furthermore, constraining the secondary structure of peptides has also been proven to enhance their biological activity. In this review, the entire process through which hydrocarbon-stapled antimicrobial peptides attain improved drug-like properties is examined. First, the essential secondary structural elements required for their activity as drugs are validated, specific residues are identified using alanine scanning, and stapling techniques are strategically incorporated at precise locations. Additionally, the mechanisms by which these structure-based stapled peptides function as AMPs are explored, providing a comprehensive and engaging discussion.

Keywords:  biomimetics; protein structure; stapled peptide

DOI:  https://doi.org/10.3390/biomimetics9090537
Angew Chem Int Ed Engl. 2024 Sep 25. e202413911

Stapled Peptides as Inhibitors of mRNA Deadenylation.

Sunit Pal, Ilja Gordijenko, Stefan Schmeing, Somarghya Biswas, Yasemin Akbulut, Raphael Gasper, Peter 't Hart.

  Therapeutic intervention targeting mRNA typically aims at reducing the levels of disease-causing sequences. Achieving the opposite effect of blocking the destruction of beneficial mRNA remains underexplored. The degradation of mRNA starts with the removal of poly(A) tails reducing their stability and translational activity which is mainly regulated by the CCR4-NOT complex. The subunit NOT9 binds various RNA binding proteins which recruit mRNA in a sequence-specific manner to the CCR4-NOT complex to promote their deadenylation. These RNA binding proteins interact with NOT9 through a helical NOT9 binding motif which we used as a starting point for development of the hydrocarbon stapled peptide NIP-2. The peptide (KD = 60.4 nM) was able to inhibit RNA-binding (IC50 = 333 nM) as well as the deadenylation activity of the CCR4-NOT complex in vitro while being cell-permeable (EC50 = 2.44 μM). A co-crystal structure of NIP-2 bound to NOT9 allowed further optimization of the peptide through point mutation leading to NIP-2-H27A-N3(KD = 122 nM) with high cell permeability (cell-permeability EC50 = 0.34 μM). The optimized peptide was able to inhibit deadenylation of target mRNAs when used in HeLa cells at a concentration of 100 μM demonstrating the feasibility of increasing mRNA stability.

Keywords:  stapled peptides * deadenylation * cell-permeability * mRNA stabilization * RNA-binding proteins

DOI:  https://doi.org/10.1002/anie.202413911
Chem Pharm Bull (Tokyo). 2024 ;72(9): 831-837

Structure Derivatization of IgG-Binding Peptides and Analysis of Their Secondary Structure by Circular Dichroism Spectroscopy.

Kyohei Muguruma, Akane Fukuda, Hayate Shida, Akihiro Taguchi, Kentaro Takayama, Atsuhiko Taniguchi, Yuji Ito, Yoshio Hayashi.

  Mid-sized cyclic peptides are a promising modality for modern drug discovery. Their larger interaction area coupled with an appropriate secondary structure is more suitable than small molecules for binding to the target protein. In this study, we conducted a structure derivatization of an immunoglobulin G (IgG)-binding peptide (15-IgBP), a β-hairpin-like cyclic peptide with a twisted β-strand and assessed the effect of the secondary structure on IgG-binding activity using circular dichroism (CD) spectra analysis. As a result, derivatization at the Ala5 and Gly9 positions affected the secondary structure of 15-IgBP, in particular the appearance of a small positive peak in the 220-240 nm region characteristic of 15-IgBP in the CD spectrum. Maintaining this peak at a moderate level may be important for the expression of IgG binding activity. We found the small methyl group at Ala5 to be crucial for retaining the preferred secondary structure; we also found Gly9 could be replaced by D-amino acids. By integrating these findings with previous results of the structure-activity relationship, we obtained four potent affinity peptides for IgG binding (Kd = 4.24-5.85 nM). Furthermore, we found the Gly9 position can be substituted for D-Lys. This is a new potential site for attaching functional units for conjugation with IgG for the preparation of homogeneous antibody-drug conjugates.

Keywords:  affinity peptide; antibody; secondary structure; structure–activity relationship

DOI:  https://doi.org/10.1248/cpb.c24-00430
Nucleic Acids Res. 2024 Sep 24. pii: gkae819. [Epub ahead of print]

Amphiphilic shuttle peptide delivers base editor ribonucleoprotein to correct the CFTR R553X mutation in well-differentiated airway epithelial cells.

Katarina Kulhankova, Anna X Cheng, Soumba Traore, Maud Auger, Mia Pelletier, Maxime Hervault, Kevin D Wells, Jonathan A Green, Addison Byrne, Benjamin Nelson, Mariana Sponchiado, Chandra Boosani, Caleb S Heffner, Kathy J Snow, Stephen A Murray, Raul A Villacreses, Michael V Rector, Nicholas D Gansemer, David A Stoltz, Chantal Allamargot, Frédéric Couture, Colin Hemez, Stéphanie Hallée, Xavier Barbeau, Mario Harvey, Coraline Lauvaux, Bruno Gaillet, Gregory A Newby, David R Liu, Paul B McCray, David Guay.

Base editing could correct nonsense mutations that cause cystic fibrosis (CF), but clinical development is limited by the lack of delivery methods that efficiently breach the barriers presented by airway epithelia. Here, we present a novel amphiphilic shuttle peptide based on the previously reported S10 peptide that substantially improved base editor ribonucleoprotein (RNP) delivery. Studies of the S10 secondary structure revealed that the alpha-helix formed by the endosomal leakage domain (ELD), but not the cell penetrating peptide (CPP), was functionally important for delivery. By isolating and extending the ELD, we created a novel shuttle peptide, termed S237. While S237 achieved lower delivery of green fluorescent protein, it outperformed S10 at Cas9 RNP delivery to cultured human airway epithelial cells and to pig airway epithelia in vivo, possibly due to its lower net charge. In well-differentiated primary human airway epithelial cell cultures, S237 achieved a 4.6-fold increase in base editor RNP delivery, correcting up to 9.4% of the cystic fibrosis transmembrane conductance regulator (CFTR) R553X allele and restoring CFTR channel function close to non-CF levels. These findings deepen the understanding of peptide-mediated delivery and offer a translational approach for base editor RNP delivery for CF airway disease.

DOI: https://doi.org/10.1093/nar/gkae819
J Biol Chem. 2024 Sep 19. pii: S0021-9258(24)02295-6. [Epub ahead of print] 107794

A yeast two-hybrid system to obtain triple-helical ligands from combinatorial random peptide libraries.

Ryo Masuda, Khine Phyu Phyu Thant, Kazuki Kawahara, Hiroya Oki, Tetsuya Kadonosono, Yuji Kobayashi, Takaki Koide.

Many bioactive proteins interact with collagen, recognizing amino acid sequences displayed on the triple helix. We report here a selection strategy to obtain triple-helical peptides that interact with the proteins from a combinatorial random library constructed in yeast cells. This system enables us to select them using the standard two-hybrid protocol, detecting interactions between triple-helical peptides and target proteins fused to the GAL4-activating and binding domains, respectively. The library was constructed having triple-helical peptides with a "host-guest" design in which host helix-stabilizing regions flanked guest random sequences. Using this system, we selected peptides that bind to pigment epithelium-derived factor (PEDF), a collagen-binding protein that shows anti-angiogenic and neurotrophic activities, from the libraries. Two-step selections from the total random library and subsequently from the second focused library yielded new PEDF-binding sequences that exhibited a comparable affinity to or more potent than that of the native PEDF-binding sequence in collagen. The obtained sequences also contained a variant of the PEDF-binding motif that did not match the known motif identified from the native collagen sequences. This combinatorial library system allows the chemical space of triple-helical peptides to be screened more widely than that found in native collagen, thus increasing the expectation of obtaining more specific and high-affinity peptides.

DOI: https://doi.org/10.1016/j.jbc.2024.107794
Lung Cancer. 2024 Sep 18. pii: S0169-5002(24)00491-4. [Epub ahead of print]196 107957

Blood-brain barrier and blood-brain tumor barrier penetrating peptide-drug conjugate as targeted therapy for the treatment of lung cancer brain metastasis.

Meng-Zhu Zheng, Zhan-Qun Yang, Sun-Li Cai, Li-Ting Zheng, Yuan Xue, Long Chen, Jian Lin.

  Lung cancer is the leading cause of cancer deaths worldwide. Brain metastasis of lung cancer, which counts for nearly 50% of late-stage lung cancer patients, is a sign of a really poor prognosis. However, the presence of blood-brain barrier (BBB) and blood-brain tumor barrier (BBTB) limits the penetration of drugs from the blood into the brain and thus restricts their accumulation in brain tumors. Systematic delivery of drugs into brain and brain tumor lesion using BBB and BBTB penetrating vehicles represents a promising strategy to overcome the BBB and BBTB limitations. Hence, we validated one of our previously identified BBB/BBTB penetrating peptide and its drug conjugate form for the treatment of lung cancer brain metastasis. With in vitro experiment, we first validated that the receptor LRP1, which mediated the peptide penetration of the BBB, was expressed on lung cancer cells and thus can be targeted by the peptide to overcome BBTB. With this delivery peptide, we constructed peptide-paclitaxel conjugate (the PDC) and in vitro validation showed that the PDC can across the BBB and efficiently kill lung cancer cells. We therefore constructed mouse lung cancer brain metastasis xenograft. In vivo anti-tumor validations showed that the PDC efficiently inhibited the proliferation of the brain resident lung cancer cells and significantly expanded the survival of the mouse xenograft, with no visible damages to the organs. Overall, our study provided potential therapeutic drugs for the treatment of lung cancer brain metastasis that may be clinically effective in the near future.

Keywords:  Blood-Brain Barrier; Blood-Brain Tumor Barrier; LRP1; Lung cancer brain metastases; Peptide-drug conjugates

DOI:  https://doi.org/10.1016/j.lungcan.2024.107957
J Control Release. 2024 Sep 25. pii: S0168-3659(24)00650-3. [Epub ahead of print]

PSMA and Sigma-1 receptor dual-targeted peptide mediates superior radionuclide imaging and therapy of prostate cancer.

Zhenyuan Huangfu, Jiangtao Yang, Juan Sun, Bin Xu, Lei Tao, Jiang Wu, Feng Wang, Guanglin Wang, Fenghua Meng, Zhiyuan Zhong.

  Radionuclide therapy, in particular peptide receptor radionuclide therapy (PRRT), has emerged as a valuable means to combat malignant tumors. The specific affinity of ACUPA peptide toward prostate-specific membrane antigen (PSMA) renders the successful development of PRRT for prostate cancer. The clinical outcome of PRRT is, however, generally challenged by moderate tumor uptake and off-target toxicity. Here, we report on a novel design of Sigma-1 receptor and PSMA dual-receptor targeted peptide (S1R/PSMA-P) for superior radionuclide imaging and therapy of prostate cancer. S1R/PSMA-P was acquired with good purity and could efficiently be labeled with 177Lu to yield 177Lu-S1R/PSMA-P with high specific activity and radiostability. Interestingly, 177Lu-S1R/PSMA-P revealed greatly enhanced affinity to LNCaP cells over single-targeted control 177Lu-PSMA-617. The single photon emission computed tomography (SPECT) imaging demonstrated exceptional uptake and retention of 177Lu-S1R/PSMA-P in LNCaP tumor, affording about 2-fold better tumor accumulation while largely reduced uptake by most normal tissues compared to 177Lu-PSMA-617. The selective uptake in LNCaP tumor was also visualized by positron emission tomography (PET) with 68Ga-S1R/PSMA-P. In accordance, a single and low dosage of 177Lu-S1R/PSMA-P at 11.1 MBq effectively suppressed tumor growth without causing apparent side effects. This dual-targeting strategy presents an appealing radionuclide therapy for malignant tumors.

Keywords:  Peptides; Prostate cancer; Radionuclide therapy; Targeted delivery; Theranostics

DOI:  https://doi.org/10.1016/j.jconrel.2024.09.040
Biomimetics (Basel). 2024 Aug 27. pii: 515. [Epub ahead of print]9(9):

Developing New Peptides and Peptide-Drug Conjugates for Targeting the FGFR2 Receptor-Expressing Tumor Cells and 3D Spheroids.

Mary A Biggs, Amrita Das, Beatriz G Goncalves, Molly E Murray, Sophia A Frantzeskos, Hannah L Hunt, Chau Ahn N Phan, Ipsita A Banerjee.

  In this work, we utilized a biomimetic approach for targeting KATO (III) tumor cells and 3D tumoroids. Specifically, the binding interactions of the bioactive short peptide sequences ACSAG (A-pep) and LPHVLTPEAGAT (L-pep) with the fibroblast growth factor receptor (FGFR2) kinase domain was investigated for the first time. Both peptides have been shown to be derived from natural resources previously. We then created a new fusion trimer peptide ACSAG-LPHVLTPEAGAT-GASCA (Trimer-pep) and investigated its binding interactions with the FGFR2 kinase domain in order to target the fibroblast growth factor receptor 2 (FGFR2), which is many overexpressed in tumor cells. Molecular docking and molecular dynamics simulation studies revealed critical interactions with the activation loop, hinge and glycine-rich loop regions of the FGFR2 kinase domain. To develop these peptides for drug delivery, DOX (Doxorubicin) conjugates of the peptides were created. Furthermore, the binding of the peptides with the kinase domain was further confirmed through surface plasmon resonance studies. Cell studies with gastric cancer cells (KATO III) revealed that the conjugates and the peptides induced higher cytotoxicity in the tumor cells compared to normal cells. Following confirmation of cytotoxicity against tumor cells, the ability of the conjugates and the peptides to penetrate 3D spheroids was investigated by evaluating their permeation in co-cultured spheroids grown with KATO (III) and colon tumor-associated fibroblasts (CAFs). Results demonstrated that Trimer-pep conjugated with DOX showed the highest permeation, while the ACSAG conjugate also demonstrated reasonable permeation of the drug. These results indicate that these peptides may be further explored and potentially utilized to create drug conjugates for targeting tumor cells expressing FGFR2 for developing therapeutics.

Keywords:  3D spheroids; biomimetic; fibroblast growth factor receptor (FGFR2); molecular docking; molecular dynamics; peptides; tumor cell targeting

DOI:  https://doi.org/10.3390/biomimetics9090515
Nat Biotechnol. 2024 Sep 25.

Multistate and functional protein design using RoseTTAFold sequence space diffusion.

Sidney Lyayuga Lisanza, Jacob Merle Gershon, Samuel W K Tipps, Jeremiah Nelson Sims, Lucas Arnoldt, Samuel J Hendel, Miriam K Simma, Ge Liu, Muna Yase, Hongwei Wu, Claire D Tharp, Xinting Li, Alex Kang, Evans Brackenbrough, Asim K Bera, Stacey Gerben, Bruce J Wittmann, Andrew C McShan, David Baker.

Protein denoising diffusion probabilistic models are used for the de novo generation of protein backbones but are limited in their ability to guide generation of proteins with sequence-specific attributes and functional properties. To overcome this limitation, we developed ProteinGenerator (PG), a sequence space diffusion model based on RoseTTAFold that simultaneously generates protein sequences and structures. Beginning from a noised sequence representation, PG generates sequence and structure pairs by iterative denoising, guided by desired sequence and structural protein attributes. We designed thermostable proteins with varying amino acid compositions and internal sequence repeats and cage bioactive peptides, such as melittin. By averaging sequence logits between diffusion trajectories with distinct structural constraints, we designed multistate parent-child protein triples in which the same sequence folds to different supersecondary structures when intact in the parent versus split into two child domains. PG design trajectories can be guided by experimental sequence-activity data, providing a general approach for integrated computational and experimental optimization of protein function.

DOI: https://doi.org/10.1038/s41587-024-02395-w
Cells. 2024 Sep 14. pii: 1549. [Epub ahead of print]13(18):

Application of Single Cell Type-Derived Spheroids Generated by Using a Hanging Drop Culture Technique in Various In Vitro Disease Models: A Narrow Review.

Hiroshi Ohguro, Megumi Watanabe, Tatsuya Sato, Nami Nishikiori, Araya Umetsu, Megumi Higashide, Toshiyuki Yano, Hiromu Suzuki, Akihiro Miyazaki, Kohichi Takada, Hisashi Uhara, Masato Furuhashi, Fumihito Hikage.

  Cell culture methods are indispensable strategies for studies in biological sciences and for drug discovery and testing. Most cell cultures have been developed using two-dimensional (2D) culture methods, but three-dimensional (3D) culture techniques enable the establishment of in vitro models that replicate various pathogenic conditions and they provide valuable insights into the pathophysiology of various diseases as well as more precise results in tests for drug efficacy. However, one difficulty in the use of 3D cultures is selection of the appropriate 3D cell culture technique for the study purpose among the various techniques ranging from the simplest single cell type-derived spheroid culture to the more sophisticated organoid cultures. In the simplest single cell type-derived spheroid cultures, there are also various scaffold-assisted methods such as hydrogel-assisted cultures, biofilm-assisted cultures, particle-assisted cultures, and magnet particle-assisted cultures, as well as non-assisted methods, such as static suspension cultures, floating cultures, and hanging drop cultures. Since each method can be differently influenced by various factors such as gravity force, buoyant force, centrifugal force, and magnetic force, in addition to non-physiological scaffolds, each method has its own advantages and disadvantages, and the methods have different suitable applications. We have been focusing on the use of a hanging drop culture method for modeling various non-cancerous and cancerous diseases because this technique is affected only by gravity force and buoyant force and is thus the simplest method among the various single cell type-derived spheroid culture methods. We have found that the biological natures of spheroids generated even by the simplest method of hanging drop cultures are completely different from those of 2D cultured cells. In this review, we focus on the biological aspects of single cell type-derived spheroid culture and its applications in in vitro models for various diseases.

Keywords:  3D spheroid culture; buoyant force; gravity force; hanging drop culture; in vitro model

DOI:  https://doi.org/10.3390/cells13181549
Angew Chem Int Ed Engl. 2024 Sep 24. e202416082

Development of DuoMYC: a synthetic cell penetrant miniprotein that efficiently inhibits the oncogenic transcription factor MYC.

Brecht D Ellenbroek, Jan Pascal Kahler, Damiano Arella, Cherina Lin, Willem Jespers, Eliane Ann-Katrin Züger, Micha Drukker, Sebastian J Pomplun.

  The master regulator transcription factor MYC is implicated in numerous human cancers, and its targeting is a long-standing challenge in drug development. MYC is a typical 'undruggable' target, with no binding pockets on its DNA binding domain and extensive intrinsically disordered regions. Rather than trying to target MYC directly with classical modalities, here we engineer synthetic miniproteins that can bind to MYC's target DNA, the enhancer box (E-Box), and potently inhibit MYC-driven transcription. We crafted the miniproteins via structure-based design and a combination of solid phase peptide synthesis and site-specific crosslinking. Our lead variant, DuoMYC, binds to E-Box DNA with high affinity (KD ~ 0.1 µM) and is able to enter cells and inhibit MYC-driven transcription with submicromolar potency (IC50 = 464 nM) as shown by reporter gene assay and confirmed by RNA sequencing. Notably, DuoMYC surpasses the efficacy of several other recently developed MYC inhibitors. Our results highlight the potential of engineered synthetic protein therapeutics for addressing challenging intracellular targets.

Keywords:  DNA-binding; Drug discovery; MYC; cell penetrating proteins; miniprotein engineering

DOI:  https://doi.org/10.1002/anie.202416082