bims-cepepe 2024-07-07 papers

bims-cepepe

Biomed News

on Cell-penetrating peptides

Issue of 2024‒07‒07
twenty-two papers selected by
Henry Lamb, Queensland University of Technology

The Influence of Disulfide, Thioacetal and Lanthionine-Bridges on the Conformation of a Macrocyclic Peptide.
A high hydrophobic moment arginine-rich peptide screened by a machine learning algorithm enhanced ADC antitumor activity.
PepExplainer: An explainable deep learning model for selection-based macrocyclic peptide bioactivity prediction and optimization.
A change in metal cation switches selectivity of a phospholipid sensor from phosphatidic acid to phosphatidylserine.
CYCLOPEp Builder: Facilitating cyclic peptide and nanotube research through a user-friendly web platform.
Enzymatic synthesis of peptide therapeutics.
Endosomolytic Peptides Enable the Cellular Delivery of Peptide Nucleic Acids.
Understanding β-strand mediated protein-protein interactions: tuning binding behaviour of intrinsically disordered sequences by backbone modification.
Discovery and Hit to Lead Optimization of Macrocyclic Peptides as Novel Tropomyosin Receptor Kinase A Antagonists.
Utilizing human cerebral organoids to model breast cancer brain metastasis in culture.
Chain-Length Dependence of Peptide-Lipid Bilayer Interaction Strength and Binding Kinetics: A Combined Theoretical and Experimental Approach.
On Demand Bioorthogonal Switching of an Antibody-Conjugated SPECT Probe to a Cytotoxic Payload: from Imaging to Therapy.
Asparagine Dependency is a Targetable Metabolic Vulnerability in TP53-Altered Castration-Resistant Prostate Cancer.
Multilayered Computational Framework for Designing Peptide Inhibitors of HVEM-LIGHT Interaction.
PowerNovo: de novo peptide sequencing via tandem mass spectrometry using an ensemble of transformer and BERT models.
Cryptic enzymatic assembly of peptides armed with β-lactone warheads.
A novel dual-release scaffold for fluorescent labels improves cyclic immunofluorescence.
Modulating lipid bilayer permeability and structure: Impact of hydrophobic chain length, C-3 hydroxyl group, and double bond in sphingosine.
Development of Peptide Paratope Mimics Derived from the Anti-ROR1 Antibody and Long-Acting Peptide-Drug Conjugates for Targeted Cancer Therapy.
Chikusetsu Saponin IVa liposomes modified with a retro-enantio peptide penetrating the blood-brain barrier to suppress pyroptosis in acute ischemic stroke rats.
CNS cell-derived exosome signatures as blood-based biomarkers of neurodegenerative diseases.
Inhibiting sphingosine 1-phosphate lyase: From efficacy to mechanism.

Chemistry. 2024 Jul 02. e202401654

The Influence of Disulfide, Thioacetal and Lanthionine-Bridges on the Conformation of a Macrocyclic Peptide.

Alethea Tabor, William Darling, Lianne Wieske, Declan Cook, Abil Aliev, Laurent Caron, Emily J Humphrys, Angelo Miguel Figueiredo, Flemming Hansen, Máté Erdélyi.

  Cyclisation of peptides by forming thioether (lanthionine), disulfide (cystine) or methylene thioacetal bridges between side chains is established as an important tool to stabilise a given structure, enhance metabolic stability and optimise both potency and selectivity. However, a systematic comparative study of the effects of differing bridging modalities on peptide conformation has not previously been carried out. In this paper, we have used the NMR deconvolution algorithm, NAMFIS, to determine the conformational ensembles, in aqueous solution, of three cyclic analogues of angiotensin(1-7), incorporating either disulfide, or non-reduceable thioether or methylene thioacetal bridges. We demonstrate that the major solution conformations are conserved between the different bridged peptides, but the distribution of conformations differs appreciably. This suggests that subtle differences in ring size and bridging structure can be exploited to fine-tune the conformational properties of cyclic peptides, which may modulate their bioactivities.

Keywords:  NAMFIS; NMR spectroscopy; conformation; cyclic peptide; lanthionine

DOI:  https://doi.org/10.1002/chem.202401654
J Pept Sci. 2024 Jul 01. e3628

A high hydrophobic moment arginine-rich peptide screened by a machine learning algorithm enhanced ADC antitumor activity.

Ruo-Long Su, Xue-Wei Cao, Jian Zhao, Fu-Jun Wang.

  Cell-penetrating peptides (CPPs) with better biomolecule delivery properties will expand their clinical applications. Using the MLCPP2.0 machine algorithm, we screened multiple candidate sequences with potential cellular uptake ability from the nuclear localization signal/nuclear export signal database and verified them through cell-penetrating fluorescent tracing experiments. A peptide (NCR) derived from the Rev protein of the caprine arthritis-encephalitis virus exhibited efficient cell-penetrating activity, delivering over four times more EGFP than the classical CPP TAT, allowing it to accumulate in lysosomes. Structural and property analysis revealed that a high hydrophobic moment and an appropriate hydrophobic region contribute to the high delivery activity of NCR. Trastuzumab emtansine (T-DM1), a HER2-targeted antibody-drug conjugate, could improve its anti-tumor activity by enhancing targeted delivery efficiency and increasing lysosomal drug delivery. This study designed a new NCR vector to non-covalently bind T-DM1 by fusing domain Z, which can specifically bind to the Fc region of immunoglobulin G and effectively deliver T-DM1 to lysosomes. MTT results showed that the domain Z-NCR vector significantly enhanced the cytotoxicity of T-DM1 against HER2-positive tumor cells while maintaining drug specificity. Our results make a useful attempt to explore the potential application of CPP as a lysosome-targeted delivery tool.

Keywords:  cell‐penetrating peptide; hydrophobic moment; machine learning; trastuzumab emtansine

DOI:  https://doi.org/10.1002/psc.3628
Eur J Med Chem. 2024 Jun 25. pii: S0223-5234(24)00508-7. [Epub ahead of print]275 116628

PepExplainer: An explainable deep learning model for selection-based macrocyclic peptide bioactivity prediction and optimization.

Silong Zhai, Yahong Tan, Cheng Zhu, Chengyun Zhang, Yan Gao, Qingyi Mao, Youming Zhang, Hongliang Duan, Yizhen Yin.

  Macrocyclic peptides possess unique features, making them highly promising as a drug modality. However, evaluating their bioactivity through wet lab experiments is generally resource-intensive and time-consuming. Despite advancements in artificial intelligence (AI) for bioactivity prediction, challenges remain due to limited data availability and the interpretability issues in deep learning models, often leading to less-than-ideal predictions. To address these challenges, we developed PepExplainer, an explainable graph neural network based on substructure mask explanation (SME). This model excels at deciphering amino acid substructures, translating macrocyclic peptides into detailed molecular graphs at the atomic level, and efficiently handling non-canonical amino acids and complex macrocyclic peptide structures. PepExplainer's effectiveness is enhanced by utilizing the correlation between peptide enrichment data from selection-based focused library and bioactivity data, and employing transfer learning to improve bioactivity predictions of macrocyclic peptides against IL-17C/IL-17 RE interaction. Additionally, PepExplainer underwent further validation for bioactivity prediction using an additional set of thirteen newly synthesized macrocyclic peptides. Moreover, it enabled the optimization of the IC50 of a macrocyclic peptide, reducing it from 15 nM to 5.6 nM based on the contribution score provided by PepExplainer. This achievement underscores PepExplainer's skill in deciphering complex molecular patterns, highlighting its potential to accelerate the discovery and optimization of macrocyclic peptides.

Keywords:  Bioactivity prediction; Graph neural network (GNN); Machine learning (ML); Macrocyclic peptide; Optimization; Structure-activity relationship (SAR)

DOI:  https://doi.org/10.1016/j.ejmech.2024.116628
Org Biomol Chem. 2024 Jul 03.

A change in metal cation switches selectivity of a phospholipid sensor from phosphatidic acid to phosphatidylserine.

Stephen M Butler, Bilge Ercan, Jingyao You, Luke P Schulz, Katrina A Jolliffe.

Phosphatidic acid and phosphatidylserine are anionic phospholipids with emerging signalling roles in cells. Determination of how phosphatidic acid and phosphatidylserine change location and quantity in cells over time requires selective fluorescent sensors that can distinguish these two anionic phospholipids. However, the design of such synthetic sensors that can selectively bind and respond to a single phospholipid within the complex membrane milieu remains challenging. In this work, we present a simple and robust strategy to control the selectivity of synthetic sensors for phosphatidic acid and phosphatidylserine. By changing the coordination metal of a dipicolylamine (DPA) ligand from Zn(II) to Ni(II) on the same synthetic sensor with a peptide backbone, we achieve a complete switch in selectivity from phosphatidic acid to phosphatidylserine in model lipid membranes. Furthermore, this strategy was largely unaffected by the choice and the position of the fluorophores. We envision that this strategy will provide a platform for the rational design of targeted synthetic phospholipid sensors to probe plasma and intracellular membranes.

DOI: https://doi.org/10.1039/d4ob00418c
Comput Struct Biotechnol J. 2024 Dec;25 91-94

CYCLOPEp Builder: Facilitating cyclic peptide and nanotube research through a user-friendly web platform.

Alfonso Cabezón, Fabián Suárez-Lestón, Juan R Granja, Ángel Piñeiro, Rebeca Garcia-Fandino.

  The study of cyclic peptides (CPs) and self-assembling cyclic peptide nanotubes (SCPNs) is pivotal in advancing applications in diverse fields such as biomedicine, nanoelectronics, and catalysis. Recognizing the limitations in the experimental study of these molecules, this article introduces CYCLOPEp Builder, a comprehensive web-based application designed to facilitate the design, simulation, and visualization of CPs and SCPNs. The tool is engineered to generate molecular topologies, essential for conducting Molecular Dynamics simulations that span All-Atom to Coarse-Grain resolutions. CYCLOPEp Builder's user-friendly interface simplifies the complex process of molecular modeling, providing researchers with the ability to readily construct CPs and SCPNs. The platform is versatile, equipped with various force fields, and capable of producing structures ranging from individual CPs to complex SCPNs with different sequences, offering parallel and antiparallel orientations among them. By enhancing the capacity for detailed visualization of molecular assemblies, CYCLOPEp Builder improves the understanding of CP and SCPN molecular interactions. This tool is a step forward in democratizing access to sophisticated simulations, offering an invaluable resource to the scientific community engaged in the exploration of supramolecular structures. CYCLOPEp is accessible at http://cyclopep.com/.

Keywords:  Builder; Cyclic peptides; Molecular; Molecular dynamics simulations; Self-assembling cyclic peptide nanotubes (SCPNs); Supramolecular structures

DOI:  https://doi.org/10.1016/j.csbj.2024.05.044
Nat Chem Biol. 2024 Jul 02.

Enzymatic synthesis of peptide therapeutics.

DOI: https://doi.org/10.1038/s41589-024-01658-6
bioRxiv. 2024 Jun 18. pii: 2024.06.18.599558. [Epub ahead of print]

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 L17ER 4 are highly efficacious delivery vehicles. Co-treatment of a PNA with low micromolar L17E or L17ER 4 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/10.1101/2024.06.18.599558
Chem Sci. 2024 Jul 03. 15(26): 10237-10245

Understanding β-strand mediated protein-protein interactions: tuning binding behaviour of intrinsically disordered sequences by backbone modification.

Emma E Cawood, Emily Baker, Thomas A Edwards, Derek N Woolfson, Theodoros K Karamanos, Andrew J Wilson.

A significant challenge in chemical biology is to understand and modulate protein-protein interactions (PPIs). Given that many PPIs involve a folded protein domain and a peptide sequence that is intrinsically disordered in isolation, peptides represent powerful tools to understand PPIs. Using the interaction between small ubiquitin-like modifier (SUMO) and SUMO-interacting motifs (SIMs), here we show that N-methylation of the peptide backbone can effectively restrict accessible peptide conformations, predisposing them for protein recognition. Backbone N-methylation in appropriate locations results in faster target binding, and thus higher affinity, as shown by relaxation-based NMR experiments and computational analysis. We show that such higher affinities occur as a consequence of an increase in the energy of the unbound state, and a reduction in the entropic contribution to the binding and activation energies. Thus, backbone N-methylation may represent a useful modification within the peptidomimetic toolbox to probe β-strand mediated interactions.

DOI: https://doi.org/10.1039/d4sc02240h
J Med Chem. 2024 Jul 01.

Discovery and Hit to Lead Optimization of Macrocyclic Peptides as Novel Tropomyosin Receptor Kinase A Antagonists.

Toru Yamada, Kousuke Mihara, Taichi Ueda, Daisuke Yamauchi, Masaya Shimizu, Azusa Ando, Kei Mayumi, Zenzaburo Nakata, Hidenori Mikamiyama.

Tropomyosin receptor kinases (Trks) are receptor tyrosine kinases activated by neurotrophic factors, called neurotrophins. Among them, TrkA interacts with the nerve growth factor (NGF), which leads to pain induction. mRNA-display screening was carried out to discover a hit compound 2, which inhibits protein-protein interactions between TrkA and NGF. Subsequent structure optimization improving phosphorylation inhibitory activity and serum stability was pursued using a unique process that took advantage of the peptide being synthesized by translation from mRNA. This gave peptide 19, which showed an analgesic effect in a rat incisional pain model. The peptides described here can serve as a new class of analgesics, and the structure optimization methods reported provide a strategy for discovering new peptide drugs.

DOI: https://doi.org/10.1021/acs.jmedchem.4c00715
Breast Cancer Res. 2024 Jul 01. 26(1): 108

Utilizing human cerebral organoids to model breast cancer brain metastasis in culture.

Chenran Wang, Aarti Nagayach, Harsh Patel, Lan Dao, Hui Zhu, Amanda R Wasylishen, Yanbo Fan, Ady Kendler, Ziyuan Guo.

  BACKGROUND: Metastasis, the spread, and growth of malignant cells at secondary sites within a patient's body, accounts for over 90% of cancer-related mortality. Breast cancer is the most common tumor type diagnosed and the leading cause of cancer lethality in women in the United States. It is estimated that 10-16% breast cancer patients will have brain metastasis. Current therapies to treat patients with breast cancer brain metastasis (BCBM) remain palliative. This is largely due to our limited understanding of the fundamental molecular and cellular mechanisms through which BCBM progresses, which represents a critical barrier for the development of efficient therapies for affected breast cancer patients.METHODS: Previous research in BCBM relied on co-culture assays of tumor cells with rodent neural cells or rodent brain slice ex vivo. Given the need to overcome the obstacle for human-relevant host to study cell-cell communication in BCBM, we generated human embryonic stem cell-derived cerebral organoids to co-culture with human breast cancer cell lines. We used MDA-MB-231 and its brain metastatic derivate MDA-MB-231 Br-EGFP, other cell lines of MCF-7, HCC-1806, and SUM159PT. We leveraged this novel 3D co-culture platform to investigate the crosstalk of human breast cancer cells with neural cells in cerebral organoid.
RESULTS: We found that MDA-MB-231 and SUM159PT breast cancer cells formed tumor colonies in human cerebral organoids. Moreover, MDA-MB-231 Br-EGFP cells showed increased capacity to invade and expand in human cerebral organoids.
CONCLUSIONS: Our co-culture model has demonstrated a remarkable capacity to discern the brain metastatic ability of human breast cancer cells in cerebral organoids. The generation of BCBM-like structures in organoid will facilitate the study of human tumor microenvironment in culture.

Keywords:  Brain metastasis; Breast cancer; Cell-cell communication; Cerebral organoids; Neural cells; Tumor microenvironment

DOI:  https://doi.org/10.1186/s13058-024-01865-y
Langmuir. 2024 Jul 04.

Chain-Length Dependence of Peptide-Lipid Bilayer Interaction Strength and Binding Kinetics: A Combined Theoretical and Experimental Approach.

Ryan S Smith, Dylan R Weaver, Gavin M King, Ioan Kosztin.

Physical interactions between polypeptide chains and lipid membranes underlie critical cellular processes. Yet, despite fundamental importance, key mechanistic aspects of these interactions remain elusive. Bulk experiments have revealed a linear relationship between free energy and peptide chain length in a model system, but does this linearity extend to the interaction strength and to the kinetics of lipid binding? To address these questions, we utilized a combination of coarse-grained molecular dynamics (CG MD) simulations, analytical modeling, and atomic force microscopy (AFM)-based single molecule force spectroscopy. Following previous bulk experiments, we focused on interactions between short hydrophobic peptides (WLn, n = 1, ..., 5) with 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) bilayers, a simple system that probes peptide primary structure effects. Potentials of mean force extracted from CG MD recapitulated the linearity of free energy with the chain length. Simulation results were quantitatively connected to bulk biochemical experiments via a single scaling factor of order unity, corroborating the methodology. Additionally, CG MD revealed an increase in the distance to the transition state, a result that weakens the dependence of the dissociation force on the peptide chain length. AFM experiments elucidated rupture force distributions and, through modeling, intrinsic dissociation rates. Taken together, the analysis indicates a rupture force plateau in the WLn-POPC system, suggesting that the final rupture event involves the last 2 or 3 residues. In contrast, the linear dependence on chain length was preserved in the intrinsic dissociation rate. This study advances the understanding of peptide-lipid interactions and provides potentially useful insights for the design of peptides with tailored membrane-interacting properties.

DOI: https://doi.org/10.1021/acs.langmuir.4c01218
J Am Chem Soc. 2024 Jul 01.

On Demand Bioorthogonal Switching of an Antibody-Conjugated SPECT Probe to a Cytotoxic Payload: from Imaging to Therapy.

Pragya Adhikari, Guangmin Li, MaryAnn Go, Danielle Mandikian, Hanine Rafidi, Carl Ng, Sagana Anifa, Kevin Johnson, Linda Bao, Hilda Hernandez Barry, Rebecca Rowntree, Nicholas Agard, Cong Wu, Kang-Jye Chou, Donglu Zhang, Katherine R Kozak, Thomas H Pillow, Gail D Lewis, Shang-Fan Yu, C Andrew Boswell, Jack D Sadowsky.

Antibody-drug conjugates (ADCs) for the treatment of cancer aim to achieve selective delivery of a cytotoxic payload to tumor cells while sparing normal tissue. In vivo, multiple tumor-dependent and -independent processes act on ADCs and their released payloads to impact tumor-versus-normal delivery, often resulting in a poor therapeutic window. An ADC with a labeled payload would make synchronous correlations between distribution and tissue-specific pharmacological effects possible, empowering preclinical and clinical efforts to improve tumor-selective delivery; however, few methods to label small molecules without destroying their pharmacological activity exist. Herein, we present a bioorthogonal switch approach that allows a radiolabel attached to an ADC payload to be removed tracelessly at will. We exemplify this approach with a potent DNA-damaging agent, the pyrrolobenzodiazepine (PBD) dimer, delivered as an antibody conjugate targeted to lung tumor cells. The radiometal chelating group, DOTA, was attached via a novel trans-cyclooctene (TCO)-caged self-immolative para-aminobenzyl (PAB) linker to the PBD, stably attenuating payload activity and allowing tracking of biodistribution in tumor-bearing mice via SPECT-CT imaging (live) or gamma counting (post-mortem). Following TCO-PAB-DOTA reaction with tetrazines optimized for extra- and intracellular reactivity, the label was removed to reveal the unmodified PBD dimer capable of inducing potent tumor cell killing in vitro and in mouse xenografts. The switchable antibody radio-drug conjugate (ArDC) we describe integrates, but decouples, the two functions of a theranostic given that it can serve as a diagnostic for payload delivery in the labeled state, but can be switched on demand to a therapeutic agent (an ADC).

DOI: https://doi.org/10.1021/jacs.4c03529
Cancer Res. 2024 Jul 03.

Asparagine Dependency is a Targetable Metabolic Vulnerability in TP53-Altered Castration-Resistant Prostate Cancer.

Young A Yoo, Songhua Quan, William Yang, Qianyu Guo, Yara Rodríguez, Zachary R Chalmers, Mary F Dufficy, Barbara Lackie, Vinay Sagar, Kenji Unno, Mihai I Truica, Navdeep S Chandel, Sarki A Abdulkadir.

The TP53 tumor suppressor is frequently altered in lethal, castration-resistant prostate cancer (CRPC). However, to date there are no effective treatments that specifically target TP53 alterations. Using transcriptomic and metabolomic analyses, we showed here that TP53-altered prostate cancer (PCa) exhibits an increased dependency on asparagine and overexpresses asparagine synthetase (ASNS), the enzyme catalyzing the synthesis of asparagine. Mechanistically, loss or mutation of TP53 transcriptionally activated ASNS expression, directly as well as via mTORC1-mediated ATF4 induction, driving de novo asparagine biosynthesis to support CRPC growth. TP53-altered CRPC cells were sensitive to asparagine restriction by knockdown of ASNS or L-asparaginase treatment to deplete the intracellular and extracellular sources of asparagine, respectively, and cell viability was rescued by asparagine addition. Notably, pharmacological inhibition of intracellular asparagine biosynthesis using a glutaminase inhibitor and depletion of extracellular asparagine with L-asparaginase significantly reduced asparagine production and effectively impaired CRPC growth. This study highlights the significance of ASNS-mediated metabolic adaptation as a synthetic vulnerability in CRPC with TP53 alterations, providing a rationale for targeting asparagine production to treat these lethal prostate cancers.

DOI: https://doi.org/10.1158/0008-5472.CAN-23-2910
J Phys Chem B. 2024 Jul 03.

Multilayered Computational Framework for Designing Peptide Inhibitors of HVEM-LIGHT Interaction.

Piotr Ciura, Pamela Smardz, Marta Spodzieja, Adam K Sieradzan, Pawel Krupa.

The herpesvirus entry mediator (HVEM) and its ligand LIGHT play crucial roles in immune system regulation, including T-cell proliferation, B-cell differentiation, and immunoglobulin secretion. However, excessive T-cell activation can lead to chronic inflammation and autoimmune diseases. Thus, inhibiting the HVEM-LIGHT interaction emerges as a promising therapeutic strategy for these conditions and in preventing adverse reactions in organ transplantation. This study focused on designing peptide inhibitors, targeting the HVEM-LIGHT interaction, using molecular dynamics (MD) simulations of 65 peptides derived from HVEM. These peptides varied in length and disulfide-bond configurations, crucial for their interaction with the LIGHT trimer. By simulating 31 HVEM domain variants, including the full-length protein, we assessed conformational changes upon LIGHT binding to understand the influence of HVEM segments and disulfide bonds on the binding mechanism. Employing multitrajectory microsecond-scale, all-atom MD simulations and molecular mechanics with generalized Born and surface area (MM-GBSA) binding energy estimation, we identified promising CRD2 domain variants with high LIGHT affinity. Notably, point mutations in these variants led to a peptide with a single disulfide bond (C58-C73) and a K54E substitution, exhibiting the highest binding affinity. The importance of the CRD2 domain and Cys58-Cys73 disulfide bond for interrupting HVEM-LIGHT interaction was further supported by analyzing truncated CRD2 variants, demonstrating similar binding strengths and mechanisms. Further investigations into the binding mechanism utilized steered MD simulations at various pulling speeds and umbrella sampling to estimate the energy profile of HVEM-based inhibitors with LIGHT. These comprehensive analyses revealed key interactions and different binding mechanisms, highlighting the increased binding affinity of selected peptide variants. Experimental circular dichroism techniques confirmed the structural properties of these variants. This study not only advances our understanding of the molecular basis of HVEM-LIGHT interactions but also provides a foundation for developing novel therapeutic strategies for immune-related disorders. Furthermore, it sets a gold standard for peptide inhibitor design in drug development due to its systematic approach.

DOI: https://doi.org/10.1021/acs.jpcb.4c02255
Sci Rep. 2024 07 01. 14(1): 15000

PowerNovo: de novo peptide sequencing via tandem mass spectrometry using an ensemble of transformer and BERT models.

Denis V Petrovskiy, Kirill S Nikolsky, Liudmila I Kulikova, Vladimir R Rudnev, Tatiana V Butkova, Kristina A Malsagova, Arthur T Kopylov, Anna L Kaysheva.

The primary objective of analyzing the data obtained in a mass spectrometry-based proteomic experiment is peptide and protein identification, or correct assignment of the tandem mass spectrum to one amino acid sequence. Comparison of empirical fragment spectra with the theoretical predicted one or matching with the collected spectra library are commonly accepted strategies of proteins identification and defining of their amino acid sequences. Although these approaches are widely used and are appreciably efficient for the well-characterized model organisms or measured proteins, they cannot detect novel peptide sequences that have not been previously annotated or are rare. This study presents PowerNovo tool for de novo sequencing of proteins using tandem mass spectra acquired in a variety of types of mass analyzers and different fragmentation techniques. PowerNovo involves an ensemble of models for peptide sequencing: model for detecting regularities in tandem mass spectra, precursors, and fragment ions and a natural language processing model, which has a function of peptide sequence quality assessment and helps with reconstruction of noisy sequences. The results of testing showed that the performance of PowerNovo is comparable and even better than widely utilized PointNovo, DeepNovo, Casanovo, and Novor packages. Also, PowerNovo provides complete cycle of processing (pipeline) of mass spectrometry data and, along with predicting the peptide sequence, involves the peptide assembly and protein inference blocks.

DOI: https://doi.org/10.1038/s41598-024-65861-0
Nat Chem Biol. 2024 Jul 01.

Cryptic enzymatic assembly of peptides armed with β-lactone warheads.

Guangcai Xu, Daniele Torri, Sebastian Cuesta-Hoyos, Deepanjan Panda, Luke R L Yates, Rémi Zallot, Kehan Bian, Dongxu Jia, Andreea I Iorgu, Colin Levy, Sarah A Shepherd, Jason Micklefield.

Nature has evolved biosynthetic pathways to molecules possessing reactive warheads that inspired the development of many therapeutic agents, including penicillin antibiotics. Peptides armed with electrophilic warheads have proven to be particularly effective covalent inhibitors, providing essential antimicrobial, antiviral and anticancer agents. Here we provide a full characterization of the pathways that nature deploys to assemble peptides with β-lactone warheads, which are potent proteasome inhibitors with promising anticancer activity. Warhead assembly involves a three-step cryptic methylation sequence, which is likely required to reduce unfavorable electrostatic interactions during the sterically demanding β-lactonization. Amide-bond synthetase and adenosine triphosphate (ATP)-grasp enzymes couple amino acids to the β-lactone warhead, generating the bioactive peptide products. After reconstituting the entire pathway to β-lactone peptides in vitro, we go on to deliver a diverse range of analogs through enzymatic cascade reactions. Our approach is more efficient and cleaner than the synthetic methods currently used to produce clinically important warhead-containing peptides.

DOI: https://doi.org/10.1038/s41589-024-01657-7
RSC Chem Biol. 2024 Jul 03. 5(7): 684-690

A novel dual-release scaffold for fluorescent labels improves cyclic immunofluorescence.

Thorge Reiber, Christian Dose, Dmytro A Yushchenko.

Cyclic immunofluorescence is a powerful method to generate high-content imaging datasets for investigating cell biology and developing therapies. This method relies on fluorescent labels that determine the quality of immunofluorescence and the maximum number of staining cycles that can be performed. Here we present a novel fluorescent labelling strategy, based on antibodies conjugated to a scaffold containing two distinct sites for enzymatic cleavage of fluorophores. The scaffold is composed of a dextran decorated with short ssDNA that upon hybridization with complementary dye-modified oligos result in fluorescent molecules. The developed fluorescent labels exhibit specific staining and remarkable brightness in flow cytometry and fluorescence microscopy. We showed that the combination of DNase-mediated degradation of DNA and dextranse-mediated degradation of the dextran as two complementary enzymatic release mechanisms in one molecule, improves signal erasure from labelled epitopes. We envision that such dual-release labels with high brightness and efficient and specific erasure will advance multiplexed cyclic immunofluorescence approaches and thereby will contribute to gaining new insights in cell biology.

DOI: https://doi.org/10.1039/d4cb00007b
J Colloid Interface Sci. 2024 Jun 24. pii: S0021-9797(24)01420-6. [Epub ahead of print]674 513-526

Modulating lipid bilayer permeability and structure: Impact of hydrophobic chain length, C-3 hydroxyl group, and double bond in sphingosine.

Yonghang Mu, Zi Wang, Linhua Song, Kun Ma, Yao Chen, Peixun Li, Zifeng Yan.

  Sphingosine, an amphiphilic molecule, plays a pivotal role as the core structure of sphingolipids, essential constituents of cell membranes. Its unique capability to enhance the permeability of lipid membranes profoundly influences crucial life processes. The molecular structure of sphingosine dictates its mode of entry into lipid bilayers and governs its interactions with lipids, thereby determining membrane permeability. However, the incomplete elucidation of the relationship between the molecular structure of sphingosine and the permeability of lipid membranes persists due to challenges associated with synthesizing sphingosine molecules. A series of sphingosine-derived molecules, featuring diverse hydrophobic chain lengths and distinct headgroup structure, were meticulously designed and successfully synthesized. These molecules were employed to investigate the permeability of large unilamellar vesicles, functioning as model lipid bilayers. With a decrease in the hydrophobic chain length of sphingosine from C15 to C11, the transient leakage ratio of vesicle contents escalated from ∼ 13 % to ∼ 28 %. Although the presence of double bond did not exert a pronounced influence on transient leakage, it significantly affected the continuous leakage ratio. Conversely, modifying the chirality of the C-3 hydroxyl group gives the opposite result. Notably, methylation at the C-3 hydroxyl significantly elevates transient leakage while suppressing the continuous leakage ratio. Additionally, sphingosines that significantly affect vesicle permeability tend to have a more pronounced impact on cell viability. Throughout this leakage process, the charge state of sphingosine-derived molecule aggregates in the solution emerged as a pivotal factor influencing vesicle permeability. Fluorescence lifetime experiments further revealed discernible variations in the effect of sphingosine molecular structure on the mobility of hydrophobic regions within lipid bilayers. These observed distinctions emphasize the impact of molecular structure on intermolecular interactions, extending to the microscopic architecture of membranes, and underscore the significance of subtle alterations in molecular structure and their associated aggregation behaviors in governing membrane permeability.

Keywords:  Aggregation behaviors; Membrane permeability; Molecular structure; Sphingosine

DOI:  https://doi.org/10.1016/j.jcis.2024.06.171
J Med Chem. 2024 Jun 29.

Development of Peptide Paratope Mimics Derived from the Anti-ROR1 Antibody and Long-Acting Peptide-Drug Conjugates for Targeted Cancer Therapy.

Yang Zhang, Yiqing Fan, Shuyu Liu, Yonghui Guan, Jiale Wan, Qiang Ren, Jialing Wang, Li Zhong, Zhipeng Hu, Wei Shi, Hai Qian.

Antibody-based targeted therapy in cancer faces a challenge due to uneven antibody distribution in solid tumors, hindering effective drug delivery. We addressed this by developing peptide mimetics with nanomolar-range affinity for Receptor Tyrosine Kinase-Like Orphan Receptor 1 (ROR1) using computational methods. These peptides showed both specific targeting and deep penetration in vitro and in vivo. Additionally, we created peptide-drug conjugates (PDCs) by linking targeting peptides to toxin drugs via various linkers and enhancing their in vivo half-life with fatty side chains for albumin binding. The antitumor candidate II-3 displayed exceptional affinity (KD = 1.72 × 10-9 M), internalization efficiency, anticancer potency (IC50 = 0.015 ± 0.002 μM), and pharmacokinetics (t1/2 = 2.6 h), showcasing a rational approach for designing PDCs with favorable tissue distribution and strong tumor penetration.

DOI: https://doi.org/10.1021/acs.jmedchem.4c00511
J Nanobiotechnology. 2024 Jul 04. 22(1): 393

Chikusetsu Saponin IVa liposomes modified with a retro-enantio peptide penetrating the blood-brain barrier to suppress pyroptosis in acute ischemic stroke rats.

Yitong Liang, Tingting Fan, Min Bai, Na Cui, Wangting Li, Jingwen Wang, Yue Guan.

  BACKGROUND: The therapeutic strategies for acute ischemic stroke were faced with substantial constraints, emphasizing the necessity to safeguard neuronal cells during cerebral ischemia to reduce neurological impairments and enhance recovery outcomes. Despite its potential as a neuroprotective agent in stroke treatment, Chikusetsu saponin IVa encounters numerous challenges in clinical application.RESULT: Brain-targeted liposomes modified with THRre peptides showed substantial uptake by bEnd. 3 and PC-12 cells and demonstrated the ability to cross an in vitro blood-brain barrier model, subsequently accumulating in PC-12 cells. In vivo, they could significantly accumulate in rat brain. Treatment with C-IVa-LPs-THRre notably reduced the expression of proteins in the P2RX7/NLRP3/Caspase-1 pathway and inflammatory factors. This was evidenced by decreased cerebral infarct size and improved neurological function in MCAO rats.
CONCLUSION: The findings indicate that C-IVa-LPs-THRre could serve as a promising strategy for targeting cerebral ischemia. This approach enhances drug concentration in the brain, mitigates pyroptosis, and improves the neuroinflammatory response associated with stroke.

Keywords:  Acute ischemic stroke; Chikusetsu saponin IVa; Liposomes; Retro-enantio peptide; THRre-peptide

DOI:  https://doi.org/10.1186/s12951-024-02641-y
Front Neurosci. 2024 ;18 1426700

CNS cell-derived exosome signatures as blood-based biomarkers of neurodegenerative diseases.

Calvin Park, Jonathan S Weerakkody, Raphael Schneider, Sheng Miao, David Pitt.

  Molecular biomarkers require the reproducible capture of disease-associated changes and are ideally sensitive, specific and accessible with minimal invasiveness to patients. Exosomes are a subtype of extracellular vesicles that have gained attention as potential biomarkers. They are released by all cell types and carry molecular cargo that reflects the functional state of the cells of origin. These characteristics make them an attractive means of measuring disease-related processes within the central nervous system (CNS), as they cross the blood-brain barrier (BBB) and can be captured in peripheral blood. In this review, we discuss recent progress made toward identifying blood-based protein and RNA biomarkers of several neurodegenerative diseases from circulating, CNS cell-derived exosomes. Given the lack of standardized methodology for exosome isolation and characterization, we discuss the challenges of capturing and quantifying the molecular content of exosome populations from blood for translation to clinical use.

Keywords:  blood-based biomarkers; central nervous system; exosomes; extracellular vesicles; neurodegeneration; neuroinflammation

DOI:  https://doi.org/10.3389/fnins.2024.1426700
Neurobiol Dis. 2024 Jun 30. pii: S0969-9961(24)00185-2. [Epub ahead of print]199 106585

Inhibiting sphingosine 1-phosphate lyase: From efficacy to mechanism.

Nelson George, Junhua Xiao.

  Sphingosine-1 phosphate (S1P) is a lipid metabolite regulating diverse biological processes, including proliferation, differentiation, migration, and apoptosis, highlighting its physiological and therapeutic significance. Current S1P-based therapeutic approaches primarily focus on modulating the downstream signalling via targeting S1P receptors, however, this is challenged by incomplete receptor internalisation. Sphingosine-1-phosphate lyase (SPL) is a highly conserved enzyme that "gatekeeps" the final step of S1P degradation. Cognisant of the complex ligand and receptor interaction and dynamic metabolic networks, the selective modulation of SPL activity presents a new opportunity to regulate S1P biosynthesis and reveal its role in various systems. Over the past decade, an evolving effort has been made to identify new molecules that could block SPL activity in vitro or in vivo. This review focuses on summarising the current understanding of the reported SPL inhibitors identified through various screening approaches, discussing their efficacy in diverse model systems and the possible mechanism of action. Whilst effective modulation of S1P levels via inhibiting SPL is feasible, the specificity of those inhibitors remains inconclusive, presenting a clear challenge for future implications. Yet, none of the currently available SPL inhibitors is proven effective in elevating S1P levels within the central nervous system. This review article embraces future research focusing on investigating selective SPL inhibitors with high potency and possibly blood-brain-barrier permeability, which would aid the development of new S1P-based therapeutics for neurological disorders.

Keywords:  Glial cells; Inhibitor; Neuron; S1P; SPL; Sphingosine 1 phosphate; Sphingosine 1 phosphate lyase

DOI:  https://doi.org/10.1016/j.nbd.2024.106585