bims-cepepe 2024-12-15 papers

bims-cepepe

Biomed News

on Cell-penetrating peptides

Issue of 2024–12–15
nine papers selected by
Henry Lamb, Queensland University of Technology

Deep mutational scanning-guided design of a high-affinity helix-loop-helix peptide targeting G-CSF receptor.
Diversity-oriented synthesis of second generation guanidinium-rich transporters toward cell-selective penetration.
Optimized Construction of a Yeast SICLOPPS Library for Unbiased In Vivo Selection of Cyclic Peptides.
Modular Total Synthesis and Antimycobacterial Activity of Rufomycins.
Navigating cancer therapy: Harnessing the power of peptide-drug conjugates as precision delivery vehicles.
Identification of cell type-specific cell-penetrating peptides through in vivo phage display leveraged by next generation sequencing.
Constrained β-Hairpins Targeting the EphA4 Ligand Binding Domain.
In silico screening of protein-binding peptides with an application to developing peptide inhibitors against antibiotic resistance.
Scaffold-free development of multicellular tumor spheroids with spatial characterization of structure and metabolic radial profiles.

Bioorg Med Chem Lett. 2024 Dec 10. pii: S0960-894X(24)00473-6. [Epub ahead of print] 130071

Deep mutational scanning-guided design of a high-affinity helix-loop-helix peptide targeting G-CSF receptor.

Masataka Michigami, Yuka Kanata, Chang Iou Ven, Ayana Oshima, Asako Yamaguchi-Nomoto, Takayoshi Kinoshita, Takatsugu Hirokawa, Ikuo Fujii.

  At present, mid-sized binding peptides have emerged as a new class of drug modalities. We have de novo designed a helix-loop-helix (HLH) peptide (MW: ∼4,500), constructed phage-displayed libraries, and screened the libraries against a variety of disease-related proteins to successfully obtain molecular-targeting HLH peptides. The next essential step in developing HLH peptides into therapeutics involves affinity engineering to optimize binding affinity and specificity. Here, we demonstrate deep mutational scanning to improve binding affinity over 1000-fold for an HLH peptide (P8-2KA; KD = 380 nM) targeting granulocyte colony-stimulation factor receptor (G-CSFR). Site-saturation mutagenesis on the two helices was performed to produce a phage-displayed library that was screened against G-CSFR. The DNA sequences of mutants from the unselected and selected phage libraries were analyzed with next-generation sequencing. The enrichment ratio of each mutant was calculated from the sequencing data to identify beneficial mutations for G-CSFR binding. Grafting of the five beneficial mutations on P8-2KA dramatically increased the binding affinity (KD = 16 nM), while cyclization of the HLH peptide with an intramolecular disulfide bond further increased binding affinity for G-CSFR (KD = 0.18 nM). The combined strategy of phage-displayed library selection and deep mutational scanning-guided design generated high-affinity HLH peptides, emphasizing the potential of molecular-targeting HLH peptides as a new drug modality that serves as an alternative to antibodies.

Keywords:  Deep mutational scanning; G-CSFR; Helix–loop–helix peptide; Phage display

DOI:  https://doi.org/10.1016/j.bmcl.2024.130071
Bioorg Chem. 2024 Dec 09. pii: S0045-2068(24)00946-5. [Epub ahead of print]154 108041

Diversity-oriented synthesis of second generation guanidinium-rich transporters toward cell-selective penetration.

Duc Tai Nguyen, Michael Desgagné, Andréanne Laniel, Christine Lavoie, Pierre-Luc Boudreault.

  Cell-penetrating peptides (CPPs) hold significant promise for intracellular delivery of various cargo molecules such as therapeutics. However, the lack of selectivity remains a critical challenge and limits the clinical application of CPPs. Using an automated peptide synthesizer, we generated a diversity-oriented library of 256 peptidomimetics containing four modified peptoid guanidine-bearing monomers incorporated alternatively with four α-amino acids. These α-amino acids were chosen to enhance lipophilic interactions with the cell membrane (Phe, 2Nal) or to bear pH-sensitive properties (His), which could enhance cancer cell selectivity. The synthesized library exhibits selective internalization, with an average selectivity index (SI) of 1.49 for HeLa cells in comparison to non-cancerous HEK293 cells. Compounds 155 and 187, containing three His residues and either Phe or 2Nal, show high cellular uptake in HeLa cells (64.6% and 75.7%, respectively) and possess an SI of 2.7 and 2.9, respectively, at the tested dose of 5 μM. Altogether, these findings highlight the use of diversity-oriented library synthesis to identify cell-permeable candidates as well as their potential for targeted cellular delivery and enhanced specificity.

Keywords:  Cell-penetrating peptides; Guanidinium-rich transporters; Peptidomimetic; Solid-phase synthesis; Structure-activity relationships

DOI:  https://doi.org/10.1016/j.bioorg.2024.108041
Biochemistry. 2024 Dec 06.

Optimized Construction of a Yeast SICLOPPS Library for Unbiased In Vivo Selection of Cyclic Peptides.

Nanna Birkmose, Emilie U Frydendahl, Charlotte R Knudsen.

DNA-encoded libraries hold great potential for discovering small, cyclized peptides with drug potential. Split-intein circular ligation of peptides and proteins (SICLOPPS) is a well-established method for in vivo selection of cyclic peptides targeting specific intracellular components. However, the method has mainly been used in prokaryotic cells. In contrast, selection studies performed directly in eukaryotic cells allow for the identification of cyclic peptides promoting a functional outcome, without the need to define a specific cellular target. Here, we report the construction of a Saccharomyces cerevisiae-specific SICLOPPS library of 80 million members, via careful optimization of several steps to increase the size of the library. Individual library members were shown to be correctly expressed and processed in yeast. High-throughput sequencing was conducted on the randomized primer used for library construction and the pure yeast SICLOPPS library isolated from Escherichia coli. A distinct guanine insertion bias was observed in the peptide-encoding, randomized sequence, which was primarily attributed to the degenerate primer used to introduce the randomized sequence. Moreover, high-throughput sequencing was performed on the library before and after the induction of cyclic peptide expression in yeast. Importantly, expression of the SICLOPPS library in S. cerevisiae caused only a marginal further sequence bias. Our work paves the way for selection studies using a large and diverse library to identify cyclic peptides of therapeutic interest that promote a specific phenotypic outcome in eukaryotic organisms, with yeast representing a beneficial model system due to its high transformation efficiency.

DOI: https://doi.org/10.1021/acs.biochem.4c00013
Org Lett. 2024 Dec 09.

Modular Total Synthesis and Antimycobacterial Activity of Rufomycins.

Max J Bedding, Bryton C Forster, Andrew M Giltrap, Maxwell T Stevens, Leo Corcilius, Warwick J Britton, Richard J Payne.

The rufomycins are a family of nonribosomal cyclic peptides isolated from the deep sea-dwelling Streptomyces atratus. Herein, we describe the total synthesis of six congeners in the rufomycin family. Synthesis was achieved through a modular solid-phase strategy, incorporating synthetic nonproteinogenic amino acids: l-2-amino-4-hexenoic acid, tert-prenyl-l-tryptophan (and related (S)-epoxide), and N-methyl-δ-hydroxy-l-leucine. Following macrolactamization, these peptides were further diversified through late-stage oxidation and secondary cyclization to furnish a library of six synthetic natural products. Rufomycins 4 and 22, bearing an unusual 6-hydroxypiperidin-2-one structural motif, exhibited impressive activity against the virulent H37Rv strain of Mycobacterium tuberculosis (MIC50 = 350-670 nM).

DOI: https://doi.org/10.1021/acs.orglett.4c04163
Eur J Med Chem. 2024 Dec 07. pii: S0223-5234(24)01013-4. [Epub ahead of print]283 117131

Navigating cancer therapy: Harnessing the power of peptide-drug conjugates as precision delivery vehicles.

Bulbul Sagar, Sarthak Gupta, Sarvesh Kumar Verma, Y Veera Manohara Reddy, Shefali Shukla.

  Cancer treatment is a formidable challenge due to the adverse effects associated with non-selective therapies like chemotherapy and radiotherapy. This review article primarily centers on the application of Peptide-Drug Conjugates (PDCs) for delivering cancer treatment. PDCs represent a promising class of precision medicines, harnessing the unique attributes of peptides in conjunction with non-peptide components. The covalent linking of peptides and drugs through specialized connectors characterizes PDCs. These constructs play a pivotal role in delivering drugs directly to tumor sites with high precision. PDCs encompass three pivotal components: a targeting ligand, a cytotoxic ligand, and a carefully chosen linker. The selection of these elements is crucial to maximize the efficiency of PDCs. PDCs offer a multitude of advantages over conventional drug molecules, including enhanced specificity, reduced off-target effects, and an improved therapeutic profile. The peptide component within PDCs can be customized to specifically adhere to disease-specific receptors or biomarkers, facilitating targeted drug delivery and accumulation in afflicted cells or tissues. This targeted approach enables the controlled release of therapeutic payloads at the localized site, resulting in heightened effectiveness and minimized systemic toxicity. Diverse linker strategies are employed to ensure the stable connection between the peptide and non-peptide components, ensuring controlled drug release at the desired location of action. The peptides utilized in these treatments encompass cell-penetrating peptides, peptides designed to target tumor cells, and those aimed at the nucleus of cancer cells. While certain clinical trials have been conducted, and some PDCs are currently in use for cancer treatment, it's essential to acknowledge that PDCs have their limitations, such as low stability in plasma, fast elimination and limited oral bioavailability. Ongoing research endeavors seek to surmount these challenges and further establish PDCs as potent agents for cancer treatment. This review sheds light on recent advancements in the design, delivery, and applications of PDCs, while also highlighting the prevailing challenges and charting a path for future research directions.

Keywords:  Antitumor activity; Cancer therapy; Cytotoxic agents; Drug delivery system; Linkers; Peptide-drug conjugate; Tumor-targeting peptides

DOI:  https://doi.org/10.1016/j.ejmech.2024.117131
Biomed Pharmacother. 2024 Dec 12. pii: S0753-3322(24)01626-3. [Epub ahead of print]182 117740

Identification of cell type-specific cell-penetrating peptides through in vivo phage display leveraged by next generation sequencing.

Franziska Kohl, Oliver Laufkötter, Mike Firth, Luc Krimpenfort, Priyanka Mangla, Mohammadhassan Ansarizadeh, Gökçe Geylan, Lauri Eklund, Leonardo De Maria, Lars Jakobsson, John Wiseman.

  Vascular anomalies (VA) refer to abnormal blood or lymphatic vessel architecture, most often as a result of dysregulated growth. Venous malformations (VM), a subgroup of VAs, are triggered by activating mutations in the Angiopoietin/TIE2-PI3K/AKT/mTOR signaling pathway with TIE2 L914F (gene name TEK) being one of the most frequent mutations in patients with VMs. Although systemic targeting of the overactivated pathway is possible, it would be a therapeutic advantage to restrict treatment to only the affected lesions. To identify peptides with potential selective binding to TIE2 L914F lesions we applied in vivo phage display to TIE2 L914F-overexpressing endothelial cells (ECs) in a subcutaneous matrigel xenograft mouse model of VMs. By panning for lesion-targeting phages in combination with subcellular fractionation, a screen for cell-penetrating candidate phages was established. Employing Next Generation Sequencing (NGS) and a refined bioinformatic analysis we were able to identify many novel cell-penetrating peptides (CPPs). To pinpoint the most selective and viable CCP candidates a hierarchical clustering algorithm was utilized. This method aggregated CPPs with highly similar sequences into a small number of clusters from which consensus sequences could be derived. Selected candidate CPPs exhibited uptake in TIE2 L914F-expressing human umbilical vein endothelial cells (HUVEC) in culture and were able to deliver siRNA into these cells. In conclusion, our NGS bioinformatic-supported approach led to the identification of novel and selective CPPs capable of transporting a siRNA cargo into targeted cells.

Keywords:  Bioinformatic analysis; Cell-penetrating peptides; In vivo phage display; Next generation sequencing; Vascular anomalies

DOI:  https://doi.org/10.1016/j.biopha.2024.117740
J Med Chem. 2024 Dec 10.

Constrained β-Hairpins Targeting the EphA4 Ligand Binding Domain.

Anne Marie Prentiss, Carlo Baggio, James Pagett, Anna O Kulinich, Iryna M Ethell, Kendall Muzzarelli, Zahra Assar, Maurizio Pellecchia.

The activity of the receptor tyrosine kinase EphA4 has been implicated in several pathologies including oncology (gastric and pancreatic cancers) and neurodegenerative diseases (amyotrophic lateral sclerosis and Alzheimer's disease). However, advances in validating EphA4 as a possible drug target have been limited by the lack of suitable pharmacological inhibitors. Recently, we reported on the design of potent EphA4 agonistic agents targeting its ligand binding domain (LBD). Based on previous studies with a phage display cyclic peptide inhibitor, we designed a β-hairpin mimetic with high affinity for EphA4-LBD. These agents hold great promise for further validation and development of EphA4-based therapeutics. Moreover, our studies introduce a possible strategy for the design of constrained β-hairpin peptides.

DOI: https://doi.org/10.1021/acs.jmedchem.4c02286
PNAS Nexus. 2024 Dec;3(12): pgae541

In silico screening of protein-binding peptides with an application to developing peptide inhibitors against antibiotic resistance.

Xianjin Xu, Wei-Ling Kao, Allison Wang, Hsin-Jou Lee, Rui Duan, Hannah Holmes, Fabio Gallazzi, Juan Ji, Hongmin Sun, Xiao Heng, Xiaoqin Zou.

  The field of therapeutic peptides is experiencing a surge, fueled by their advantageous features. These include predictable metabolism, enhanced safety profile, high selectivity, and reduced off-target effects compared with small-molecule drugs. Despite progress in addressing limitations associated with peptide drugs, a significant bottleneck remains: the absence of a large-scale in silico screening method for a given protein target structure. Such methods have proven invaluable in accelerating small-molecule drug discovery. The high flexibility of peptide structures and the large diversity of peptide sequences greatly hinder the development of urgently needed computational methods. Here, we report a method called MDockPeP2_VS to address these challenges. It integrates molecular docking with structural conservation between protein folding and protein-peptide binding. Briefly, we discovered that when the interfacial residues are conserved, a sequence fragment derived from a monomeric protein exhibits a high propensity to bind a target protein with a similar conformation. This valuable insight significantly reduces the search space for peptide conformations, resulting in a substantial reduction in computational time and making in silico peptide screening practical. We applied MDockPeP2_VS to develop peptide inhibitors targeting the TEM-1 β-lactamase of Escherichia coli, a key mechanism behind antibiotic resistance in gram-negative bacteria. Among the top 10 peptides selected from in silico screening, TF7 (KTYLAQAAATG) showed significant inhibition of β-lactamase activity with a K i value of 1.37 ± 0.37 µM. This fully automated, large-scale structure-based in silico peptide screening software is available for free download at https://zougrouptoolkit.missouri.edu/mdockpep2_vs/download.html.

Keywords:  antibiotic resistance; in silico peptide screening; molecular docking; peptide drug discovery; protein–peptide interactions

DOI:  https://doi.org/10.1093/pnasnexus/pgae541
In Vitro Model. 2024 ;3(2-3): 91-108

Scaffold-free development of multicellular tumor spheroids with spatial characterization of structure and metabolic radial profiles.

Shelby N Bess, Gaven K Smart, Matthew J Igoe, Timothy J Muldoon.

   Purpose: In vitro assays are essential for studying cellular biology, but traditional monolayer cultures fail to replicate the complex three-dimensional (3D) interactions of cells in living organisms. 3D culture systems offer a more accurate reflection of the cellular microenvironment. However, 3D cultures require robust and unique methods of characterization.
Methods: The goal of this study was to create a 3D spheroid model using cancer cells and macrophages, and to demonstrate a custom image analysis program to assess structural and metabolic changes across spheroid microregions.
Results: Structural characterization shows that cells at the necrotic core show high normalized fluorescence intensities of CD206 (M2 macrophages), cellular apoptosis (cleaved caspase-3, CC3), and hypoxia (HIF-1α and HIF-2α) compared to the proliferative edge, which shows high normalized fluorescence intensities of CD80 (M1 macrophages) and cellular proliferation (Ki67). Metabolic characterization was performed using multiphoton microscopy and fluorescence lifetime imaging (FLIM). Results show that the mean NADH lifetime at the necrotic core (1.011 ± 0.086 ns) was lower than that at the proliferative edge (1.105 ± 0.077 ns). The opposite trend is shown in the A1/A2 ratio (necrotic core: 4.864 ± 0.753; proliferative edge: 4.250 ± 0.432).
Conclusion: Overall, the results of this study show that 3D multicellular spheroid models can provide a reliable solution for studying tumor biology, allowing for the evaluation of discrete changes across all spheroid microregions.

Keywords:  Autofluorescence; Fluorescence microscopy; Macrophage; Metabolism; Multicellular spheroids

DOI:  https://doi.org/10.1007/s44164-024-00074-3