bims-cepepe 2023-09-17 papers

bims-cepepe

Biomed News

on Cell-penetrating peptides

Issue of 2023‒09‒17
eighteen papers selected by
Henry Lamb, Queensland University of Technology

Design and evaluation of α-helix-based peptide inhibitors for blocking PD-1/PD-L1 interaction.
Identification of sodium channel toxins from marine cone snails of the subgenera Textilia and Afonsoconus.
Design, synthesis and bioactivity investigation of peptide-camptothecin conjugates as anticancer agents with a potential to overcome drug resistance.
pyPept: a python library to generate atomistic 2D and 3D representations of peptides.
Fluorophore-Tagged Poly-Lysine RAFT Agents: Controlled Synthesis of Trackable Cell-Penetrating Peptide-Polymers.
Display Selection of a Hybrid Foldamer-Peptide Macrocycle.
A Versatile Elastin-Like Carrier for Bioactive Antimicrobial Peptide Production and Delivery.
Engineering Proteins for Cell Entry.
Investigation of Peptides for Molecular Recognition of C-Reactive Protein-Theoretical and Experimental Studies.
Use of tree-based machine learning methods to screen affinitive peptides based on docking data.
A high-throughput microfluidic mechanoporation platform to enable intracellular delivery of cyclic peptides in cell-based assays.
Targeting NANOG and FAK via Cx26-derived cell-penetrating peptides in triple-negative breast cancer.
Unveiling the interaction between DNA G-quadruplexes and RG-rich peptides.
Potential role of the cell-penetrating peptide-conjugated soluble N-ethylmaleimide-sensitive factor attachment protein receptor motif of vesicle-associated membrane protein 2-patterned peptide in novel cosmeceutical skin product development.
Analysis of a Novel Peptide That Is Capable of Inhibiting the Enzymatic Activity of the Protein Kinase A Catalytic Subunit-Like Protein from Trypanosoma equiperdum.
Facts and prospects of peptide in targeted therapy and immune regulation against triple-negative breast cancer.
Stearyl palmitate a multi-target inhibitor against breast cancer: in-silico, in-vitro & in-vivo approach.
Multiplexed experimental strategies for fragment library screening against challenging drug targets using SPR biosensors.

Int J Biol Macromol. 2023 Sep 09. pii: S0141-8130(23)03708-X. [Epub ahead of print]253(Pt 2): 126811

Design and evaluation of α-helix-based peptide inhibitors for blocking PD-1/PD-L1 interaction.

Mengjie Rui, Wen Zhang, Ke Mi, Hairong Ni, Wei Ji, Xuefei Yu, Jiangjiang Qin, Chunlai Feng.

  The current research in tumor immunotherapy indicates that blocking the protein-protein interaction (PPI) between PD-1 and its ligand, PD-L1, may be one of the most effective treatments for cancer patients. The α-helix is a common elements of protein secondary structure and is often involved in protein interaction. Thus, α-helix-based peptides could mimic proteins involved in such interactions and are also capable of modulating PPI in vivo. In this study, starting from a potential α-helix-rich protein, we designed a series of α-helix-based peptide candidates to block PD-1/PD-L1 interaction. These candidates were first screened using molecular docking and molecular dynamics simulations, and then their capacities to inhibit PD-1/PD-L1 interactions and to restore antitumor immune activities were investigated using the HTRF assay, SPR assay, cellular co-culture experiments and animal model experiments. Two peptides exhibited the best anti-tumor effects and the strong ability to restore the immunity of tumor-infiltrating T-cells. Further D-amino acid substitution was employed to improve the serum stability of peptide candidate, making the intravenous administration easier while maintaining the therapeutic efficacy. The resultant peptides showed promise as checkpoint inhibitors for application in tumor immunotherapy. These findings suggested that our strategy for developing peptides starting from an α-helical structure could be used in the design of bioactive inhibitors to potential block protein-protein interactions.

Keywords:  ApoA-1 mimetic peptides; Cancer immunotherapy; PD-1/PD-L1 checkpoint; Peptide inhibitor; Virtual screening

DOI:  https://doi.org/10.1016/j.ijbiomac.2023.126811
Cell Mol Life Sci. 2023 Sep 09. 80(10): 287

Identification of sodium channel toxins from marine cone snails of the subgenera Textilia and Afonsoconus.

Kirsten L McMahon, Henrik O'Brien, Christina I Schroeder, Jennifer R Deuis, Dhananjeyan Venkatachalam, Di Huang, Brad R Green, Pradip K Bandyopadhyay, Qing Li, Mark Yandell, Helena Safavi-Hemami, Baldomero M Olivera, Irina Vetter, Samuel D Robinson.

  Voltage-gated sodium (NaV) channels are transmembrane proteins that play a critical role in electrical signaling in the nervous system and other excitable tissues. µ-Conotoxins are peptide toxins from the venoms of marine cone snails (genus Conus) that block NaV channels with nanomolar potency. Most species of the subgenera Textilia and Afonsoconus are difficult to acquire; therefore, their venoms have yet to be comprehensively interrogated for µ-conotoxins. The goal of this study was to find new µ-conotoxins from species of the subgenera Textilia and Afonsoconus and investigate their selectivity at human NaV channels. Using RNA-seq of the venom gland of Conus (Textilia) bullatus, we identified 12 µ-conotoxin (or µ-conotoxin-like) sequences. Based on these sequences we designed primers which we used to identify additional µ-conotoxin sequences from DNA extracted from historical specimens of species from Textilia and Afonsoconus. We synthesized six of these µ-conotoxins and tested their activity on human NaV1.1-NaV1.8. Five of the six synthetic peptides were potent blockers of human NaV channels. Of these, two peptides (BuIIIB and BuIIIE) were potent blockers of hNaV1.3. Three of the peptides (BuIIIB, BuIIIE and AdIIIA) had submicromolar activity at hNaV1.7. This study serves as an example of the identification of new peptide toxins from historical DNA and provides new insights into structure-activity relationships of µ-conotoxins with activity at hNaV1.3 and hNaV1.7.

Keywords:  Conotoxin; Historical DNA; Voltage-gated sodium channel; µ-conotoxin

DOI:  https://doi.org/10.1007/s00018-023-04935-0
Int J Pharm. 2023 Sep 09. pii: S0378-5173(23)00823-2. [Epub ahead of print]645 123402

Design, synthesis and bioactivity investigation of peptide-camptothecin conjugates as anticancer agents with a potential to overcome drug resistance.

Chenyu Zhang, Honglan Zhong, Xiang Li, Zhenjian Xing, Jiaqi Liu, Rui Yu, Xin Deng.

  Camptothecin (CPT) is a natural plant alkaloid from Camptotheca that exhibits a potent anticancer activity. However, its continued utilization is hindered by drawbacks such as low water solubility and restricted tumor selectivity. Cationic anticancer peptides (CAPs) are generally soluble in water, and exhibit favorable selectivity against malignant cells. In previous study, we have reported a CAP termed KM8-Aib present conspicuous selective anticancer effect. Thus, it is postulated conjugating KM8-Aib with CPT might be a plausible approach to improve the defects of CPT. A series of peptide-CPT conjugates were synthesized and subjected to biological evaluation. Among these compounds, Kb-CC07 displayed the highest selective activity against a set of cancer cell lines including drug-resistant cells, showing the IC50 values in the 0.11-1.01 μM range which is 1.9-22.6 times better than that of CPT, and a wide therapeutic index of 124.5 (vs 5.3 for CPT). The water solubility of Kb-CC07 was also improved by ∼ 100 fold compared with CPT. Further investigation unraveled that Kb-CC07 could effectively penetrate across plasma membranes and delivered more CPT molecules into cancer cells, overcoming the drug-resistance result from efflux drug transporters on tumor surface. In vivo experiments supported that Kb-CC07 has excellent in vivo antiproliferative activity against drug-resistant tumors over CPT (tumor growth inhibition of 98.2% and 37.5% for Kb-CC07 and CPT, respectively, at 5 μmol·kg-1), and prompts CPT accumulation in tumor tissue rather than normal organs, thus producing limited toxicities. To sum up, coupling therapeutic agents to CAPs would be a potential strategy to conquer the shortcomings of anticancer drugs. Additionally, Kb-CC07 is suggested to be a promising anticancer candidate deserving further investigation.

Keywords:  Anticancer; Camptothecin (CPT); Drug resistance; Peptide-drug conjugates; Selectivity

DOI:  https://doi.org/10.1016/j.ijpharm.2023.123402
J Cheminform. 2023 Sep 12. 15(1): 79

pyPept: a python library to generate atomistic 2D and 3D representations of peptides.

Rodrigo Ochoa, J B Brown, Thomas Fox.

  We present pyPept, a set of executables and underlying python-language classes to easily create, manipulate, and analyze peptide molecules using the FASTA, HELM, or recently-developed BILN notations. The framework enables the analysis of both pure proteinogenic peptides as well as those with non-natural amino acids, including support to assemble a customizable monomer library, without requiring programming. From line notations, a peptide is transformed into a molecular graph for 2D depiction tasks, the calculation of physicochemical properties, and other systematic analyses or processing pipelines. The package includes a module to rapidly generate approximate peptide conformers by incorporating secondary structure restraints either given by the user or predicted via pyPept, and a wrapper tool is also provided to automate the generation and output of 2D and 3D representations of a peptide directly from the line notation. HELM and BILN notations that include circular, branched, or stapled peptides are fully supported, eliminating errors in structure creation that are prone during manual drawing and connecting. The framework and common workflows followed in pyPept are described together with illustrative examples. pyPept has been released at: https://github.com/Boehringer-Ingelheim/pyPept .

Keywords:  BILN; Cheminformatics; Conformer; Molecule depiction; Peptide; Python; RDKit

DOI:  https://doi.org/10.1186/s13321-023-00748-2
ACS Macro Lett. 2023 Sep 11. 1280-1285

Fluorophore-Tagged Poly-Lysine RAFT Agents: Controlled Synthesis of Trackable Cell-Penetrating Peptide-Polymers.

Paige A Shaw, Maxime Klausen, Annamaria Lilienkampf, Mark Bradley.

The conjugation of a fluorophore and a variety of cell-penetrating peptides onto a RAFT agent allowed for the synthesis of polymers of defined sizes with quantifiable cell-uptake. Each peptide-RAFT agent was used to polymerize acrylamide, acrylate, and styrene monomers to form high or low molecular weight polymers (here 50 or 7.5 kDa) with the peptide having no influence on the RAFT agent's control. The incorporation of a single fluorophore per polymer chain allowed cellular analysis of the uptake of the size-specific peptide-polymers via flow cytometry and confocal microscopy. The cell-penetrating peptides had a direct effect on the efficiency of polymer uptake for both high and low molecular weight polymers, demonstrating the versatility of the strategy. These "all-in-one", synthetically accessible RAFT agents allow highly controlled preparation of synthetic peptide-polymer conjugates and subsequent quantification of their delivery into cells.

DOI: https://doi.org/10.1021/acsmacrolett.3c00460
Angew Chem Int Ed Engl. 2023 Sep 14. e202308408

Display Selection of a Hybrid Foldamer-Peptide Macrocycle.

Sebastian Dengler, Ryan T Howard, Vasily Morozov, Christos Tsiamantas, Wei-En Huang, Zhiwei Liu, Christopher Dobrzanski, Vojislava Pophristic, Sophie Brameyer, Céline Douat, Hiroaki Suga, Ivan Huc.

  Expanding the chemical diversity of peptide macrocycle libraries for display selection is desirable to improve their potential at binding biomolecular targets. We now have implemented a considerable expansion through a large aromatic helical foldamer inclusion. A helical aromatic foldamer was identified that undergoes flexizyme-mediated tRNA acylation and is capable of initiating ribosomal translation with yields sufficiently high to perform an mRNA display selection of macrocyclic foldamer-peptide hybrids. A hybrid macrocyle nanomolar binder to the C-lobe of the E6AP HECT domain was selected that showed a highly converged peptide sequence. A crystal structure and molecular dynamics simulations revealed that both the peptide and foldamer are helical in an intriguing reciprocal stapling fashion. The strong residue convergence could be rationalized based on their involvement in specific interactions with the target protein. The foldamer stabilizes the peptide helix through stapling and through contacts with key residues. These results altogether represent a significant extension of the chemical space amenable to display selection and highlight possible benefits of inserting an aromatic foldamer into a peptide macrocycle for the purpose of protein recognition.

Keywords:  Display selection; Foldamer; Peptide; Protein recognition; macrocycle

DOI:  https://doi.org/10.1002/anie.202308408
Macromol Biosci. 2023 Sep 12. e2300236

A Versatile Elastin-Like Carrier for Bioactive Antimicrobial Peptide Production and Delivery.

Laura ColominaAlfaro, Paola Sist, Silvia Marchesan, Ranieri Urbani, Artemis Stamboulis, Antonella Bandiera.

  Elastin-like polypeptides are biotechnological protein and peptide carriers that offer a vast scope of applicability. This work aims to build a model for the expression of antimicrobial peptides by genetically engineering the Human Elastin-like Polypeptide platform developed in our lab. The well-characterized antimicrobial peptide indolicidin is selected as an example of antimicrobial domain for the recombinant fusion at the C-terminus of the carrier. The fusion construct has been designed to allow the release of the antimicrobial domain. The expression product has been purified and its physicochemical and antimicrobial properties has been characterized. Taking advantage of the self-assembling and matrix-forming properties of the recombinant biopolymer, the materials that are obtained have been evaluated for the antimicrobial activity toward bacterial-strain models. Our approach represents a cost-effective strategy for the production of smart components and materials endowed with antimicrobial capacity triggered by external stimuli. This article is protected by copyright. All rights reserved.

Keywords:  antimicrobial peptide; hydrogel matrix; recombinant biopolymers; smart materials

DOI:  https://doi.org/10.1002/mabi.202300236
Mol Pharm. 2023 Sep 14.

Engineering Proteins for Cell Entry.

Xiaoyu Feng, Ruilong Chang, Haichao Zhu, Yifan Yang, Yue Ji, Dingkang Liu, Hai Qin, Jun Yin, Haibo Rong.

  Proteins are essential for life, as they participate in all vital processes in the body. In the past decade, delivery of active proteins to specific cells and organs has attracted increasing interest. However, most proteins cannot enter the cytoplasm due to the cell membrane acting as a natural barrier. To overcome this challenge, various proteins have been engineered to acquire cell-penetrating capacity by mimicking or modifying natural shuttling proteins. In this review, we provide an overview of the different types of engineered cell-penetrating proteins such as cell-penetrating peptides, supercharged proteins, receptor-binding proteins, and bacterial toxins. We also discuss some strategies for improving endosomal escape such as pore formation, the proton sponge effect, and hijacking intracellular trafficking pathways. Finally, we introduce some novel methods and technologies for designing and detecting engineered cell-penetrating proteins.

Keywords:  endosomal escape; intracellular protein delivery; protein engineering

DOI:  https://doi.org/10.1021/acs.molpharmaceut.3c00467
Anal Chem. 2023 Sep 11.

Investigation of Peptides for Molecular Recognition of C-Reactive Protein-Theoretical and Experimental Studies.

Katarzyna Szot-Karpińska, Patryk Kudła, Urszula Orzeł, Magdalena Narajczyk, Martin Jönsson-Niedziółka, Barbara Pałys, Sławomir Filipek, Andreas Ebner, Joanna Niedziółka-Jönsson.

We investigate the interactions between C-reactive protein (CRP) and new CRP-binding peptide materials using experimental (biological and physicochemical) methods with the support of theoretical simulations (computational modeling analysis). Three specific CRP-binding peptides (P2, P3, and P9) derived from an M13 bacteriophage have been identified using phage-display technology. The binding efficiency of the peptides exposed on phages toward the CRP protein was demonstrated via biological methods. Fibers of the selected phages/peptides interact differently due to different compositions of amino acid sequences on the exposed peptides, which was confirmed by transmission electron microscopy. Numerical and experimental studies consistently showed that the P3 peptide is the best CRP binder. A combination of theoretical and experimental methods demonstrates that identifying the best binder can be performed simply, cheaply, and fast. Such an approach has not been reported previously for peptide screening and demonstrates a new trend in science where calculations can replace or support laborious experimental techniques. Finally, the best CRP binder─the P3 peptide─was used for CRP recognition on silicate-modified indium tin oxide-coated glass electrodes. The obtained electrodes exhibit a wide range of operation (1.0-100 μg mL-1) with a detection limit (LOD = 3σ/S) of 0.34 μg mL-1. Moreover, the dissociation constant Kd of 4.2 ± 0.144 μg mL-1 (35 ± 1.2 nM) was evaluated from the change in the current. The selectivity of the obtained electrode was demonstrated in the presence of three interfering proteins. These results prove that the presented P3 peptide is a potential candidate as a receptor for CRP, which can replace specific antibodies.

DOI: https://doi.org/10.1021/acs.analchem.3c03127
Mol Inform. 2023 Sep 11.

Use of tree-based machine learning methods to screen affinitive peptides based on docking data.

Hua Feng, Fangyu Wang, Ning Li, Qian Xu, Guanming Zheng, Xuefeng Sun, Man Hu, Xuewu Li, Guangxu Xing, Gaiping Zhang.

  Screening peptides with good affinity is an important step in peptide-drug discovery. Recent advancement in computer and data science have made machine learning a useful tool in accurately affinitive-peptide screening. In current study, four different tree-based algorithms, including Classification and regression trees(CART), C5.0 decision tree (C50), Bagged CART (BAG) and Random Forest (RF), were employed to explore the relationship between experimental peptide affinities and virtual docking data, and the performance of each model was also compared in parallel. All four algorithms showed better performances on dataset pre-scaled, -centered and -PCA than other pre-processed dataset. After model re-built and hyperparameter optimization, the optimal C50 model(C50O) showed the best performances in terms of Accuracy, Kappa, Sensitivity, Specificity, F1, MCC and AUC when validated on test data and an unknown PEDV datasets evaluation (Accuracy=80.4%). BAG and RFO (the optimal RF), as two best models during training process, did not performed as expecting during in testing and unknown dataset validations. Furthermore, the high correlation of the predictions of RFO and BAG to C50O implied the high stability and robustness of their prediction. Whereas although the good performance on unknown dataset, the poor performance in test data validation and correlation analysis indicated CARTO could not be used for future data prediction. To accurately evaluate the peptide affinity, the current study firstly gave a tree-model competition on affinitive peptide prediction by using virtual docking data, which would expand the application of machine learning algorithms in studying PepPIs and benefit the development of peptide therapeutics.

Keywords:  Tree-based algorithms; affinity classification; peptides

DOI:  https://doi.org/10.1002/minf.202300143
Bioeng Transl Med. 2023 Sep;8(5): e10542

A high-throughput microfluidic mechanoporation platform to enable intracellular delivery of cyclic peptides in cell-based assays.

Stephen H Kasper, Stephanie Otten, Brian Squadroni, Cionna Orr-Terry, Yi Kuang, Lily Mussallem, Lan Ge, Lin Yan, Srinivasaraghavan Kannan, Chandra S Verma, Christopher J Brown, Charles W Johannes, David P Lane, Arun Chandramohan, Anthony W Partridge, Lee R Roberts, Hubert Josien, Alex G Therien, Erik C Hett, Bonnie J Howell, Andrea Peier, Xi Ai, Jason Cassaday.

  Cyclic peptides are poised to target historically difficult to drug intracellular protein-protein interactions, however, their general cell impermeability poses a challenge for characterizing function. Recent advances in microfluidics have enabled permeabilization of the cytoplasmic membrane by physical cell deformation (i.e., mechanoporation), resulting in intracellular delivery of impermeable macromolecules in vector- and electrophoretic-free approaches. However, the number of payloads (e.g., peptides) and/or concentrations delivered via microfluidic mechanoporation is limited by having to pre-mix cells and payloads, a manually intensive process. In this work, we show that cells are momentarily permeable (t 1/2 = 1.1-2.8 min) after microfluidic vortex shedding (μVS) and that lower molecular weight macromolecules can be cytosolically delivered upon immediate exposure after cells are processed/permeabilized. To increase the ability to screen peptides, we built a system, dispensing-microfluidic vortex shedding (DμVS), that integrates a μVS chip with inline microplate-based dispensing. To do so, we synced an electronic pressure regulator, flow sensor, on/off dispense valve, and an x-y motion platform in a software-driven feedback loop. Using this system, we were able to deliver low microliter-scale volumes of transiently mechanoporated cells to hundreds of wells on microtiter plates in just several minutes (e.g., 96-well plate filled in <2.5 min). We validated the delivery of an impermeable peptide directed at MDM2, a negative regulator of the tumor suppressor p53, using a click chemistry- and NanoBRET-based cell permeability assay in 96-well format, with robust delivery across the full plate. Furthermore, we demonstrated that DμVS could be used to identify functional, low micromolar, cellular activity of otherwise cell-inactive MDM2-binding peptides using a p53 reporter cell assay in 96- and 384-well format. Overall, DμVS can be combined with downstream cell assays to investigate intracellular target engagement in a high-throughput manner, both for improving structure-activity relationship efforts and for early proof-of-biology of non-optimized peptide (or potentially other macromolecular) tools.

Keywords:  automation; cell‐based assays; cyclic peptides; intracellular delivery; microfluidics; protein–protein interactions

DOI:  https://doi.org/10.1002/btm2.10542
Mol Cancer Ther. 2023 Sep 13.

Targeting NANOG and FAK via Cx26-derived cell-penetrating peptides in triple-negative breast cancer.

Erin E Mulkearns-Hubert, Emily Esakov Rhoades, Salma Ben-Salem, Rashmi Bharti, Nicole Hajdari, Sadie Johnson, Alex Myers, Iris Nira Smith, Smarajit Bandyopadhyay, Charis Eng, Erinn Downs, Justin D Lathia, Ofer Reizes.

Triple-negative breast cancer (TNBC) represents the most lethal and treatment-resistant breast cancer subtype with limited treatment options. We previously identified a protein complex unique to TNBC composed of the gap junction protein connexin 26 (Cx26), the pluripotency transcription factor NANOG, and focal adhesion kinase (FAK). We sought to determine whether a peptide mimetic of the interaction region of Cx26 attenuated tumor growth in pre-clinical models. We designed peptides based on Cx26 juxtamembrane domains and performed binding experiments with NANOG and FAK using surface plasmon resonance. Binding studies revealed that the Cx26 C-terminal tail and intracellular loop bound to NANOG and FAK with submicromolar-to-micromolar affinity and that a 5-amino acid sequence in the C-terminal tail of Cx26 (RYCSG) was sufficient for binding. Peptides with high affinity were engineered with a cell-penetrating antennapedia sequence and assessed in functional assays including cell proliferation, tumorsphere formation, and in vivo tumor growth, and downstream signaling changes were measured. The cell-penetrating Cx26 peptide (aCx26-pep) disrupted self-renewal while reducing nuclear FAK and NANOG and inhibiting NANOG target gene expression in TNBC cells but not luminal mammary epithelial cells. In vivo, aCx26-pep reduced tumor growth and proliferation and induced cell death. Here, we provide proof-of-concept that a Cx26 peptide-based strategy inhibits growth and alters NANOG activity specifically in TNBC, indicating the therapeutic potential of this targeting approach.

DOI: https://doi.org/10.1158/1535-7163.MCT-21-0783
Int J Biol Macromol. 2023 Sep 07. pii: S0141-8130(23)03646-2. [Epub ahead of print] 126749

Unveiling the interaction between DNA G-quadruplexes and RG-rich peptides.

Nicola Grasso, Raffaele Graziano, Simona Marzano, Federica D'Aria, Francesco Merlino, Paolo Grieco, Antonio Randazzo, Bruno Pagano, Jussara Amato.

  G-quadruplexes are non-canonical DNA secondary structures formed within guanine-rich strands that play important roles in various biological processes, including gene regulation, telomere maintenance and DNA replication. The biological functions and formation of these DNA structures are strictly controlled by several proteins that bind and stabilize or resolve them. Many G-quadruplex-binding proteins feature an arginine and glycine-rich motif known as the RGG or RG-rich motif. Although this motif plays a crucial role in the recognition of such non-canonical structures, their interaction is still poorly understood. Here, we employed a combination of several biophysical techniques to provide valuable insights into the interaction between a peptide containing an RGG motif shared by numerous human G-quadruplex-binding proteins (NIQI) and various biologically relevant G-quadruplex DNA structures with different topologies. We also shed light on the key amino acids involved in the binding process. Our findings contribute to lay the basis for the development of a new class of peptide-based G-quadruplex ligands as an alternative to small molecules. These ligands may serve as valid tools for interfering in DNA-protein interactions, with potential therapeutic applications.

Keywords:  Biophysics; DNA; G-quadruplex; Peptide; RGG motif

DOI:  https://doi.org/10.1016/j.ijbiomac.2023.126749
J Cosmet Dermatol. 2023 Sep 12.

Potential role of the cell-penetrating peptide-conjugated soluble N-ethylmaleimide-sensitive factor attachment protein receptor motif of vesicle-associated membrane protein 2-patterned peptide in novel cosmeceutical skin product development.

Hyo Jin Lee, Daehoon Kim, Hyo Jeong Choi, Suhyeok Kim, Minhee Shin, Seongsung Kwak, Dong-Kyu Lee, Won-Ho Kang.

  AIM: This study aimed to investigate and verify the effect of cell-penetrating peptide (CPP)-conjugated soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) motif of vesicle-associated membrane protein 2 (VAMP2)-patterned peptide (INCI name: Acetyl sh-Oligopeptide-26 sh-Oligopeptide-27 SP, trade name: M.Biome-BT) on improving skin function in vitro.METHODS: The cytotoxicity of CPP-conjugated SNARE motif of VAMP2-patterned peptide (CVP) was investigated using the 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) assay against B16-F10 cells and human dermal fibroblasts (HDFs) and a reconstructed skin irritation test. The anti-wrinkle activity of M.Biome-BT was determined by assessing the release of norepinephrine and dopamine in PC-12 cells via ELISA. The skin-whitening effects of CVP were assessed in B16-F10 cells by measuring the intra- and extracellular melanin contents and expression levels of melanin production-related genes, such as microphthalmia-associated transcription factor (MITF), tyrosinase (TYR), tyrosinase-related protein-1 (TRP-1), and TRP-2.
RESULTS: CVP is not cytotoxic to B16-F10 cells and HDFs, and no skin irritation was observed. CVP treatment considerably diminished K+ -induced norepinephrine and dopamine secretion compared with the non-treated control group (62% and 40%, respectively). Additionally, the inhibition ability of CVP on norepinephrine and dopamine release was comparable to that of botulinum neurotoxin type A (BoNT/A). CVP also increased intracellular melanin content in a dose-dependent manner, whereas extracellular melanin content decreased (76%-85%). However, CVP treatment did not affect the mRNA expression of MITF, TYR, TRP-1, and TRP-2. These results suggest that CVP does not inhibit melanin production; however, it may induce a whitening effect by inhibiting melanin transport.
CONCLUSIONS: Taken together, our findings indicate that CVP could be used as an active and safe cosmeceutical ingredient for antiaging applications.

Keywords:  cell-penetrating peptide; cosmetics; skin aging; soluble N-ethylmaleimide-sensitive factor attachment protein receptor; synthetic peptide

DOI:  https://doi.org/10.1111/jocd.15984
Protein J. 2023 Sep 15.

Analysis of a Novel Peptide That Is Capable of Inhibiting the Enzymatic Activity of the Protein Kinase A Catalytic Subunit-Like Protein from Trypanosoma equiperdum.

Nelson A Araujo, José Bubis.

  A 26-residue peptide possessing the αN-helix motif of the protein kinase A (PKA) regulatory subunit-like proteins from the Trypanozoom subgenera (VAP26, sequence = VAPYFEKSEDETALILKLLTYNVLFS), was shown to inhibit the enzymatic activity of the Trypanosoma equiperdum PKA catalytic subunit-like protein, in a similar manner that the mammalian heat-stable soluble PKA inhibitor known as PKI. However, VAP26 does not contain the PKI inhibitory sequence. Bioinformatics analyzes of the αN-helix motif from various Trypanozoon PKA regulatory subunit-like proteins suggested that the sequence could form favorable peptide-protein interactions of hydrophobic nature with the PKA catalytic subunit-like protein, which possibly may represent an alternative PKA inhibitory mechanism. The sequence of the αN-helix motif of the Trypanozoon proteins was shown to be highly homologous but significantly divergent from the corresponding αN-helix motifs of their Leishmania and mammalian counterparts. This sequence divergence contrasted with the proposed secondary structure of the αN-helix motif, which appeared conserved in every analyzed regulatory subunit-like protein. In silico mutation experiments at positions I234, L238 and F244 of the αN-helix motif from the Trypanozoon proteins destabilized both the specific motif and the protein. On the contrary, mutations at positions T239 and Y240 stabilized the motif and the protein. These results suggested that the αN-helix motif from the Trypanozoon proteins probably possessed a different evolutionary path than their Leishmania and mammalian counterparts. Moreover, finding stabilizing mutations indicated that new inhibitory peptides may be designed based on the αN-helix motif from the Trypanozoon PKA regulatory subunit-like proteins.

Keywords:  Analysis of sequences; Helix motif; In silico mutations; Peptide inhibitor; Protein kinase A; Trypanozoon

DOI:  https://doi.org/10.1007/s10930-023-10153-1
Front Immunol. 2023 ;14 1255820

Facts and prospects of peptide in targeted therapy and immune regulation against triple-negative breast cancer.

Yongxiu Huang, Anqi Zeng, Linjiang Song.

  Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer. Due to the lack of specific therapeutic targets, treatment options are limited, and the recurrence and metastasis rate is high, the overall survival of patients is poor. However, with the discovery of some new targets and the corresponding immune regulation after targeting these targets, TNBC has a new hope in treatment. The peptide has a simple structure, strong binding affinity, and high stability, and has great potential in targeted therapy and immune regulation against TNBC. This review will discuss how single peptides and peptide combinations target triple-negative breast cancer to exert immunomodulatory effects. Among them, single peptides target specific receptors on TNBC cells, act as decoys to target key ligands in the regulatory pathway, and target TME-related cells. The combinations of peptides work in the form of cancer vaccines, engineered exosomes, microRNAs and other immune-related molecular pathways, immune checkpoint inhibitors, chimeric antigen receptor T cells, and drug-peptide conjugates. This article is mainly dedicated to exploring new treatment methods for TNBC to improve the curative effect and prolong the survival time of patients.

Keywords:  combination; immunity; peptide; target; triple-negative breast cancer

DOI:  https://doi.org/10.3389/fimmu.2023.1255820
J Biomol Struct Dyn. 2023 Sep 10. 1-18

Stearyl palmitate a multi-target inhibitor against breast cancer: in-silico, in-vitro & in-vivo approach.

Lokesh Ravi, Ajith Kumar K, Shree Kumari G R, Harsha S, Jabin B Sam Raj, Likitha R, Prawin Chinnaiyan, David Jonnes K C, Megha J K, Dhanush Sudhakara, Musaib Shafi Dar, Yashaswini D M, Sathvik G.

  Multi-target inhibitors are currently trending in the pharmaceutical research, as they possess increased efficacy and reduced toxicity. In this study multi-target inhibitors for breast cancer are explored from a curated list of natural products, i.e. 4,670 phytochemicals belonging to 360 medicinal plants. In-silico screening of phytochemicals using SeeSAR and AutoDock Vina resulted in identification of Stearyl Palmitate as a potential drug molecule that inhibits three drug targets, i.e. HER-2, MEK-1 and PARP-1 proteins. Molecular Dynamics Simulation for 100 ns each for these three protein-ligand complexes using Desmond, Maestro platform also confirmed the prediction of multi-target inhibition by Stearyl Palmitate. Further in-vitro MTT assay demonstrated that Stearyl Palmitate has a significant IC50 value of 40 µM against MCF-7 cells and >1000 µM against L929 cells. This confirmed that Stearyl Palmitate is having selective cytotoxicity towards breast cancer cells in comparison to non-cancerous cells. Fluorescence staining and flow cytometry analysis confirmed that, Stearyl Palmitate is inducing apoptosis in MCF-7 cells at IC50 concentration. Finally, in-vivo efficacy and toxicity studies were performed using zebrafishes (Danio rerio). It was observed that the fishes treated with IC50 concentration of Stearyl Palmitate demonstrated 2x folds reduction in tumour size, while double dose resulted in 4x folds reduction in tumour size. Stearyl Palmitate did not demonstrate any toxicity or side effects in the zebrafishes. It is concluded that, Stearyl Palmitate, a phytochemical reported to be present in Althea officinalis is a potential anti-breast cancer agent, with ability to inhibit multiple targets such as HER-2, MEK-1 and PARP-2 proteins.Communicated by Ramaswamy H. Sarma.

Keywords:  Desmond; MTT assay; SeeSAR; Stearyl Palmitate; breast cancer; multi-target inhibitor

DOI:  https://doi.org/10.1080/07391102.2023.2255271
SLAS Discov. 2023 Sep 13. pii: S2472-5552(23)00067-9. [Epub ahead of print]

Multiplexed experimental strategies for fragment library screening against challenging drug targets using SPR biosensors.

Edward A FitzGerald, Darius Vagrys, Giulia Opassi, Hanna F Klein, David J Hamilton, Vladimir O Talibov, Mia Abramsson, Anna Moberg, Maria T Lindgren, Claes Holmgren, Ben Davis, Peter O'Brien, Maikel Wijtmans, Roderick E Hubbard, Iwan J P de Esch, U Helena Danielson.

  Surface plasmon resonance (SPR) biosensor methods are ideally suited for fragment-based lead discovery. However, generally applicable experimental procedures and detailed protocols are lacking, especially for structurally or physico-chemically challenging targets or when tool compounds are not available. Success depends on accounting for the features of both the target and the chemical library, purposely designing screening experiments for identification and validation of hits with desired specificity and mode-of-action, and availability of orthogonal methods capable of confirming fragment hits. The range of targets and libraries amenable to an SPR biosensor-based approach for identifying hits is considerably expanded by adopting multiplexed strategies, using multiple complementary surfaces or experimental conditions. Here we illustrate principles and multiplexed approaches for using flow-based SPR biosensor systems for screening fragment libraries of different sizes (90 and 1056 compounds) against a selection of challenging targets. It shows strategies for the identification of fragments interacting with 1) large and structurally dynamic targets, represented by acetyl choline binding protein (AChBP), a Cys-loop receptor ligand gated ion channel homologue, 2) targets in multi protein complexes, represented by lysine demethylase 1 and a corepressor (LSD1/CoREST), 3) structurally variable or unstable targets, represented by farnesyl pyrophosphate synthase (FPPS), 4) targets containing intrinsically disordered regions, represented by protein tyrosine phosphatase 1B (PTP1B), and 5) aggregation-prone proteins, represented by an engineered form of human tau (tau K18M). Practical considerations and procedures accounting for the characteristics of the proteins and libraries, and that increase robustness, sensitivity, throughput and versatility are highlighted. The study shows that the challenges for addressing these types of targets is not identification of potentially useful fragments per se, but establishing methods for their validation and evolution into leads.

Keywords:  Biophysics; Biosensors; Fragment-based drug discovery; SPR

DOI:  https://doi.org/10.1016/j.slasd.2023.09.001