bims-cepepe 2023-11-05 papers

bims-cepepe

Biomed News

on Cell-penetrating peptides

Issue of 2023–11–05
24 papers selected by
Henry Lamb, Queensland University of Technology

Cell-Surface-Retained Peptide Additives for the Cytosolic Delivery of Functional Proteins.
Cyclic Peptide Linker Design and Optimization by Molecular Dynamics Simulations.
Computational Prediction of Cyclic Peptide Structural Ensembles and Application to the Design of Keap1 Binders.
Electrochemical Nickel-Catalyzed Selective Inter- and Intramolecular Arylations of Cysteine-Containing Peptides.
Computational design of α-conotoxins to target specific nicotinic acetylcholine receptor subtypes.
Inhibition of Aurora-A/N-Myc protein-protein interaction using peptidomimetics: Understanding the role of peptide cyclization.
Neuronal membrane proteasome-derived peptides modulate NMDAR-dependent neuronal signaling to promote changes in gene expression.
Diagnostic utility of N-terminal TMPP labels for unambiguous identification of clipped sites in therapeutic proteins.
Lectin-anticancer peptide fusion demonstrates significant cancer-cell-selective cytotoxic effect and inspires the production of 'clickable' anticancer peptide in E. coli.
Targeting androgen receptor (AR) with a synthetic peptide increases apoptosis in triple negative breast cancer and AR-expressing prostate cancer cell lines.
A GPX4 non-enzymatic domain and MDM2 targeting peptide PROTAC for acute lymphoid leukemia therapy through ferroptosis induction.
Discovery of 14-3-3 PPI Stabilizers by Extension of an Amidine-Substituted Thiophene Fragment.
Peptidomimetics for CVD screened via TRADD-TRAF2 complex interface assessments.
A novel cell-permeable peptide prevents protein sumoylation and supports the mislocalization and aggregation of TDP-43.
Imaging Proteins Sensitive to Direct Fusions Using Transient Peptide-Peptide Interactions.
Disruption of blood-brain barrier: effects of HIV Tat on brain microvascular endothelial cells and tight junction proteins.
PD-1 expression on tumour-infiltrating cells is a prognostic factor for relapsed or refractory diffuse large B-cell lymphoma.
TPpred-LE: therapeutic peptide function prediction based on label embedding.
Development of a 18F-Labeled Bicyclic Peptide Targeting EphA2 for Molecular Imaging of PSMA-Negative Prostate Cancer.
Recognition and reprogramming of E3 ubiquitin ligase surfaces by α-helical peptides.
Covalent Peptide LSD1 Inhibitor Specifically Recognizes Cys360 in the Enzyme-Active Region.
Bioconjugation Techniques for Enhancing Stability and Targeting Efficiency of Protein and Peptide Therapeutics.
Triptolide Reduces MDA-MB-231 Cell Metastasis by Attenuating Epithelial-Mesenchymal Transition through the ROCK/PTEN/Akt Axis.
B2LiVe, a label-free 1D-NMR method to quantify the binding of amphitropic peptides or proteins to membrane vesicles.

J Am Chem Soc. 2023 Oct 31.

Cell-Surface-Retained Peptide Additives for the Cytosolic Delivery of Functional Proteins.

Jan Vincent V Arafiles, Jonathan Franke, Luise Franz, Jacobo Gómez-González, Kristin Kemnitz-Hassanin, Christian P R Hackenberger.

The delivery of functional proteins remains a major challenge in advancing biological and pharmaceutical sciences. Herein, we describe a powerful, simple, and highly effective strategy for the intracellular delivery of functional cargoes. Previously, we demonstrated that cell-penetrating peptide (CPP) additives equipped with electrophilic thiol-reactive moieties temporarily attach to the cellular membrane, thereby facilitating the cellular uptake of protein- and antibody-CPP cargoes through direct membrane transduction at low concentrations. Now, we hypothesize that CPP-additives with an increased retention on the cellular membrane will further enhance intracellular uptake. We discovered that adding a small hydrophobic peptide sequence to an arginine-rich electrophilic CPP-additive further improved the uptake of protein-CPP conjugates, whereas larger hydrophobic anchors showed increased cytotoxicity. Cell viability and membrane integrity measurements, structure-activity relationship studies, and quantitative evaluation of protein-CPP uptake revealed important design principles for cell-surface-retained CPP-additives. These investigations allowed us to identify a nontoxic, thiol-reactive CPP-additive containing the hydrophobic ILFF sequence, which can deliver fluorescent model proteins at low micromolar concentrations. This hydrophobic CPP-additive allowed the addition of protein cargoes for intracellular delivery after initial additive incubation. Time-lapse fluorescence microscopy and membrane tension analysis of cells treated with fluorescent ILFF-CPP-additives supported the claim of increased cell surface retention and suggested that the protein-CPP cargoes enter the cell through a mechanism involving lowered cell membrane tension. Finally, we demonstrated that our newly engineered hydrophobic CPP-additive enabled the uptake of a functional macrocyclic peptidic MDM2-inhibitor and a recombinant genome editing protein. This indicates that the developed hydrophobic CPP-additive holds promise as a tool to enhance the intracellular delivery of peptide and protein cargoes.

DOI: https://doi.org/10.1021/jacs.3c05365
J Chem Inf Model. 2023 Oct 30.

Cyclic Peptide Linker Design and Optimization by Molecular Dynamics Simulations.

Lei Yu, Stephanie A Barros, Chengzao Sun, Sandeep Somani.

Cyclic peptides are an emerging therapeutic modality that can target protein-protein interaction sites with high affinity and selectivity. A common medicinal chemistry strategy for the optimization of peptide hits is conformational stabilization through macrocyclization. We present a method based on explicit solvent enhanced sampling molecular dynamics simulations for estimating the impact of varying linker lengths and chemistry on the conformational stability of a peptide. The method is demonstrated on three cyclic peptide series that bind to proteins PCSK9, trypsin, and MDM2 adopting loop, β-sheet, and helical secondary structures. In general, the simulations show greater solution stability of the receptor-bound conformation for the higher-affinity peptides, consistent with the idea that preorganizing a ligand for binding can enhance binding affinity. The impact of the force field and sampling is discussed for one series that does not follow this trend. We have successfully applied this method to internal discovery programs to design peptides with increased potency and chemical stability.

DOI: https://doi.org/10.1021/acs.jcim.3c01359
J Chem Inf Model. 2023 Nov 02.

Computational Prediction of Cyclic Peptide Structural Ensembles and Application to the Design of Keap1 Binders.

Francini Fonseca Lopez, Jiayuan Miao, Jovan Damjanovic, Luca Bischof, Michael B Braun, Yingjie Ling, Marcus D Hartmann, Yu-Shan Lin, Joshua A Kritzer.

The Nrf2 transcription factor is a master regulator of the cellular response to oxidative stress, and Keap1 is its primary negative regulator. Activating Nrf2 by inhibiting the Nrf2-Keap1 protein-protein interaction has shown promise for treating cancer and inflammatory diseases. A loop derived from Nrf2 has been shown to inhibit Keap1 selectively, especially when cyclized, but there are no reliable design methods for predicting an optimal macrocyclization strategy. In this work, we employed all-atom, explicit-solvent molecular dynamics simulations with enhanced sampling methods to predict the relative degree of preorganization for a series of peptides cyclized with a set of bis-thioether "staples". We then correlated these predictions to experimentally measured binding affinities for Keap1 and crystal structures of the cyclic peptides bound to Keap1. This work showcases a computational method for designing cyclic peptides by simulating and comparing their entire solution-phase ensembles, providing key insights into designing cyclic peptides as selective inhibitors of protein-protein interactions.

DOI: https://doi.org/10.1021/acs.jcim.3c01337
Angew Chem Int Ed Engl. 2023 Oct 31. e202315748

Electrochemical Nickel-Catalyzed Selective Inter- and Intramolecular Arylations of Cysteine-Containing Peptides.

Linhua Shen, Olivier Monasson, Elisa Peroni, Franck Lebideau, Samir Messaoudi.

  Here we report a simple electrochemical route towards the synthesis of S-arylated peptides by a site selective coupling of peptides with aryl halides under base free conditions. This approach demonstrates the power of electrochemistry to access both highly complex peptide conjugates and to cyclic peptides.

Keywords:  Peptides Electrochemistry Nickel catalysis Stapled peptides Cyclic peptides

DOI:  https://doi.org/10.1002/anie.202315748
Chemistry. 2023 Nov 01. e202302909

Computational design of α-conotoxins to target specific nicotinic acetylcholine receptor subtypes.

Xiaosa Wu, Arik J Hone, Yen-Hua Huang, Richard J Clark, J Michael McIntosh, Quentin Kaas, David J Craik.

  Nicotinic acetylcholine receptors (nAChRs) are drug targets for neurological diseases and disorders, but selective targeting of the large number of nAChR subtypes is challenging. Marine cone snail α-conotoxins are potent blockers of nAChRs and some have been engineered to achieve subtype selectivity. This engineering effort would benefit from rapid computational methods able to predict mutational energies, but current approaches typically require high-resolution experimental structures, which are not widely available for α-conotoxin complexes. Here, we benchmarked five mutational energy prediction methods using crystallographic and mutational data on two acetylcholine binding protein/α-conotoxin systems. We developed molecular models for six nAChR subtypes in complex with five α-conotoxins that were studied through 150 substitutions. We determined that the best method was a combination of FoldX and molecular dynamics simulations, resulting in a predictive Matthews Correlation Coefficient (MCC) of 0.68 (85% accuracy). Novel α-conotoxin mutants designed using this method were successfully validated by experimental assay with improved pharmaceutical properties. This work paves the way for the rapid design of subtype-specific nAChR ligands and potentially accelerated drug development.

Keywords:  drug design; energy calculation; molecular modelling; nAChR; α-conotoxin

DOI:  https://doi.org/10.1002/chem.202302909
Chembiochem. 2023 Oct 31. e202300649

Inhibition of Aurora-A/N-Myc protein-protein interaction using peptidomimetics: Understanding the role of peptide cyclization.

Robert S Dawber, Diana Gimenez, Matthew Batchelor, Jennifer A Miles, Megan H Wright, Richard Bayliss, Andrew John Wilson.

  Using N-Myc61-89 as a starting template we showcase the systematic use of truncation and maleimide constraining to develop peptidomimetic inhibitors of the N-Myc/Aurora-A protein-protein interaction (PPI); a potential anticancer drug discovery target. The most promising of these - N-Myc73-94-N85C/G89C-mal, is shown to favour a more Aurora-A compliant binding ensemble in comparison to the linear wild-type sequence as observed through fluorescence anisotropy competition assays, circular dichroism (CD) and nuclear magnetic resonance (NMR) experiments. Further in silico investigation of this peptide in its Aurora-A bound state, by molecular dynamics (MD) simulations, imply (i) the bound conformation is more stable as a consequence of the constraint, which likely suppresses dissociation and (ii) the constraint may make further stabilizing interactions with the Aurora-A surface. Taken together this work unveils the first orthosteric N-Myc/Aurora-A inhibitor and provides useful insights on the biophysical properties and thus design of constrained peptides, an attractive therapeutic modality.

Keywords:  protein-protein interactions * N-Myc * Aurora-A kinase * constrained peptides

DOI:  https://doi.org/10.1002/cbic.202300649
Mol Biol Cell. 2023 Nov 01. mbcE23060218

Neuronal membrane proteasome-derived peptides modulate NMDAR-dependent neuronal signaling to promote changes in gene expression.

Fulya Türker, Anna Brennan, Seth S Margolis.

The neuronal membrane proteasome (NMP) degrades intracellular proteins into peptides that are released directly into the extracellular space, whereby they stimulate neurons to promote signaling mechanisms that remain unknown. Here, we demonstrate that neuronal stimulation promotes NMP activity and, subsequently, enhanced production of NMP peptides. We show that these neuronal activity-dependent NMP peptides can rapidly promote N-methyl-D-aspartate receptor (NMDAR)-dependent calcium influx in neurons. This leads to sustained phosphorylation of the well-defined stimulus-induced transcription factor, cyclic AMP response element (CRE)-binding protein (CREB). Downstream of these events, we identified changes to neuronal target genes which included increased expression of immediate early genes (e.g., Fos, Npas4, Egr4) and other genes known to have critical neuroregulatory roles. Further observations led to the discovery that NMP peptide-induced changes in gene expression is dependent on NMDARs and independent of AMPA receptors or voltage-gated sodium channels. These data demonstrate that NMP peptides are endogenous and selective activators of NMDA receptors and act as sufficient and novel stimuli within the context of neuronal activity-dependent signaling. This novel pathway is parallel to classic neuronal activity-dependent programs and points to NMP and its resulting peptides as potential modulators of neuronal function. [Media: see text] [Media: see text] [Media: see text].

DOI: https://doi.org/10.1091/mbc.E23-06-0218
Sci Rep. 2023 Oct 30. 13(1): 18602

Diagnostic utility of N-terminal TMPP labels for unambiguous identification of clipped sites in therapeutic proteins.

Harsha P Gunawardena, Meth M Jayatilake, Jeffery D Brelsford, Hirsh Nanda.

Protein therapeutics are susceptible to clipping via enzymatic and nonenzymatic mechanisms that create neo-N-termini. Typically, neo-N-termini are identified by chemical derivatization of the N-terminal amine with (N-Succinimidyloxycarbonylmethyl)tris(2,4,6-trimethoxyphenyl)phosphonium bromide (TMPP) followed by proteolysis and mass spectrometric analysis. Detection of the TMPP-labeled peptide is achieved by mapping the peptide sequence to the product ion spectrum derived from collisional activation. The site-specific localization of the TMPP tag enables unambiguous determination of the true N-terminus or neo-N-termini. In addition to backbone product ions, TMPP reporter ions at m/z 573, formed via collision-induced dissociation, can be diagnostic for the presence of a processed N-termini. However, reporter ions generated by collision-induced dissociation may be uninformative because of their low abundance. We demonstrate a novel high-throughput LC-MS method for the facile generation of the TMPP reporter ion at m/z 533 and, in some instances m/z 590, upon electron transfer dissociation. We further demonstrate the diagnostic utility of TMPP labeled peptides derived from a total cell lysate shows high degree of specificity towards selective N-terminal labeling over labeling of lysine and tyrosine and highly-diagnostic Receiver Operating Characteristic's (ROC) of TMPP reporter ions of m/z 533 and m/z 590. The abundant generation of these reporters enables subsequent MS/MS by intensity and m/z-dependent triggering of complementary ion activation modes such as collision-induced dissociation, high-energy collision dissociation, or ultraviolet photo dissociation for subsequent peptide sequencing.

DOI: https://doi.org/10.1038/s41598-023-45446-z
Protein Sci. 2023 Nov 02. e4830

Lectin-anticancer peptide fusion demonstrates significant cancer-cell-selective cytotoxic effect and inspires the production of 'clickable' anticancer peptide in E. coli.

Rajeev Pasupuleti, Sabrina Riedl, Laia Saltor Núñez, Marianna Karava, Vajinder Kumar, Robert Kourist, W Bruce Turnbull, Dagmar Zweytick, Birgit Wiltschi.

  Targeted killing of tumor cells while protecting healthy cells is the pressing priority in cancer treatment. Lectins that target a specific glycan marker abundant on cancer cells can be valuable new tools for selective cancer cell killing. The lectin shiga-like toxin 1 B subunit (Stx1B) is an example that specifically binds globotriaosylceramide (CD77 or Gb3), which is overexpressed in certain cancers. In this study, a human lactoferricin-derived synthetic retro di-peptide R-DIM-P-LF11-215 with antitumor efficacy was fused to the lectin Stx1B to selectively target and kill Gb3+ cancer cells. We produced lectin-peptide fusion proteins in E. coli, isolated them by Gb3-affinity chromatography and assessed their ability to selectively kill Gb3+ cancer cells in a Calcein AM assay. Furthermore, to expand the applications of R-DIM-P-LF11-215 in developing therapeutic bioconjugates, we labelled R-DIM-P-LF11-215 with the unique reactive non-canonical amino acid Nε -((2-azidoethoxy)carbonyl)-L-lysine (AzK) at a selected position by amber stop codon suppression. The R-DIM-P-LF11-215 20AzK and the unlabeled R-DIM-P-LF11-215 parent peptide were produced as GST-fusion proteins for soluble expression in E. coli for the first time. We purified both variants by size-exclusion chromatography and analyzed their peptide masses. Finally, a cyanin 3 fluorophore was covalently conjugated to R-DIM-P-LF11-215 20AzK by strain-promoted alkyne-azide cycloaddition. Our results showed that the recombinant lectin-peptide fusion R-DIM-P-LF11-215-Stx1B killed >99% Gb3+ HeLa cells while Gb3-negative cells were unaffected. The peptides R-DIM-P-LF11-215 and R-DIM-P-LF11-215 20AzK were produced recombinantly in E. coli in satisfactory amounts and were tested functional by cytotoxicity and cell-binding assays, respectively. This article is protected by copyright. All rights reserved.

Keywords:  Human lactoferricin derived peptide; anticancer peptide; click chemistry; fusion proteins; peptide purification; recombinant peptide production; targeted drug delivery

DOI:  https://doi.org/10.1002/pro.4830
Cancer Rep (Hoboken). 2023 Oct 30. e1922

Targeting androgen receptor (AR) with a synthetic peptide increases apoptosis in triple negative breast cancer and AR-expressing prostate cancer cell lines.

Mazdak Jamshidi, Fatemeh Keshavarzi, Sabrieh Amini, Ismail Laher, Ali Gheysarzadeh, Kambiz Davari.

   BACKGROUND: The androgen receptor (AR) has been studied as an approach to cancer therapy.
AIMS: We used human breast cancer-derived cells with high, low, and very low expression levels of AR, in addition to prostate cancer-derived LNCaP and DU-145 cells as a positive and negative controls to examine apoptosis caused by a synthetic peptide that targets ARs.
METHODS AND RESULTS: The peptide was produced to inhibit AR transactivation in breast cancer cell lines. We then measured cell viability, caspase-3 activity, and the ratio of Bax/Bcl-2. The findings indicated that the peptide (100-500 nM) in the presence of dihydrotestosterone (DHT) reduced cell growth in cells with high and low expression level of AR (p < .001), but not in cells with very low levels of AR. Treatment with 100-500 nM of peptide activated caspase-3 and increased the ratio of Bax/Bcl-2 in cells with high and low expression levels of AR. Also, increasing concentrations of the peptide (100-500 nM) reduced BrdU incorporation in the presence of DHT and promoted apoptosis in cells with high and low expression levels of AR (p < .001).
CONCLUSION: The findings indicate the peptide significantly increased apoptosis in cancer cells.

Keywords:  androgen receptor; anticancer peptide; apoptosis; breast cancer; dihydrotestosterone (DHT)

DOI:  https://doi.org/10.1002/cnr2.1922
Biochem Biophys Res Commun. 2023 Oct 20. pii: S0006-291X(23)01209-3. [Epub ahead of print]684 149125

A GPX4 non-enzymatic domain and MDM2 targeting peptide PROTAC for acute lymphoid leukemia therapy through ferroptosis induction.

Fan Niu, Runyu Yang, Hui Feng, Yi Liu, Ruimin Liu, Bohan Ma.

  Ferroptosis, an emerging form of programmed cell death, has garnered substantial attention as a potential target for cancer therapy. However, despite the potential promise, no ferroptosis-related therapies have progressed to clinical trials. Identifying disease types sensitive to ferroptosis and developing specific ferroptosis-targeting drugs are critical focal points in the field of ferroptosis-based treatment. In this study, we conducted a comprehensive database analysis and presented compelling evidence indicating a high expression of GPX4 in patients with acute lymphoblastic leukemia (ALL), significantly correlating with poor prognosis. Notably, elevated GPX4 expression is closely associated with ALL relapse, a major challenge in the treatment of this disease. Building upon these findings, we devised a novel peptide-based Proteolysis Targeting Chimeras (PROTAC) drug targeting GPX4 through computer-aided design. In contrast to existing drugs that target the conjugative enzyme active site, our design focused on a peptide drug targeting the non-active site of GPX4. Furthermore, we strategically selected MDM2, an E3 ligase highly expressed in ALL, for the PROTAC drug design. This deliberate choice amplifies the drug's effect on cancer cells while minimizing its impact on normal cells, achieving desirable selectivity for cancer cells. Leveraging nanogold delivery, we successfully facilitated intracellular action of the GPX4-targeting peptide PROTAC drug, denoted as Au-PGPD (peptide GPX4 PROTAC drug). Au-PGPD effectively induced GPX4 degradation and inhibited ALL cell proliferation. Remarkably, Au-PGPD exhibited significantly less efficacy on normal cells, underscoring the selectivity and safety of our design.

Keywords:  ALL; Ferroptosis; GPX4; MDM2; PROTAC drug; Peptide drug

DOI:  https://doi.org/10.1016/j.bbrc.2023.149125
Chembiochem. 2023 Oct 30. e202300636

Discovery of 14-3-3 PPI Stabilizers by Extension of an Amidine-Substituted Thiophene Fragment.

Qi Wu, Federica Centorrino, Xavier Guillory, Madita Wolter, Christian Ottmann, Peter J Cossar, Luc Brunsveld.

  Protein-protein interaction (PPI) modulation is a promising approach in drug discovery with the potential to expand the 'druggable' proteome and develop new therapeutic strategies. While there have been significant advancements in methodologies for developing PPI inhibitors, there is a relative scarcity of literature describing the 'bottom-up' development of PPI stabilizers (Molecular Glues). The hub protein 14-3-3 and its interactome provide an excellent platform for exploring conceptual approaches to PPI modulation, including evolution of chemical matter for Molecular Glues. In this study, we employed a fragment extension strategy to discover stabilizers for the complex of 14-3-3 protein and an Estrogen Receptor alpha-derived peptide (ERα). A focused library of analogues derived from an amidine-substituted thiophene fragment enhanced the affinity of the 14-3-3/ERα complex up to 6.2-fold. Structure-activity relationship (SAR) analysis underscored the importance of the newly added, aromatic side chain with a certain degree of rigidity. X-ray structural analysis revealed a unique intermolecular π-π stacking binding mode of the most active analogues, resulting in the simultaneous binding of two molecules to the PPI binding pocket. Notably, analogue 11 displayed selective stabilization of the 14-3-3/ERα complex.

Keywords:  14-3-3; ERα; Fragment; Molecular Glue; PPI Stabilizer

DOI:  https://doi.org/10.1002/cbic.202300636
In Silico Pharmacol. 2023 ;11(1): 28

Peptidomimetics for CVD screened via TRADD-TRAF2 complex interface assessments.

A Manikandan, S Jeevitha, Laharika Vusa.

  The main aim of this study is to screen and develop Peptidomimetics to treat atherosclerosis (AS) which is a Cardio Vascular Disease (CVD). Peptidomimetics were obtained from the protein-protein interaction interface of TRADD (Tumor necrosis factor receptor type 1-associated DEATH domain protein) and TRAF2 (TNF receptor-associated factor 2) complex. TRADD-TRAF2 interaction is critical in AS pathogenesis since it assists a series of signal transducers that activate NF-κB. Conceptually, the triggered NF-κB makes an extensive amount of nitric oxide (NO) synthesized by inducible nitric oxide synthase (iNOS), which boons the progress of AS. The examined TRADD-TRAF2 complex (PDB ID: 1F3V) and its interaction details revealed that the sequence range W11-G165 of TRADD highly interacts with TRAF2. The sequence range W11-G165 was selected for the design and preparation of the inhibitory peptide in silico. The selected sequence was mutated with the alanine scanning method to have a range of inhibitory peptides. With the help of different in silico tools, the top three, namely MIP11-25 L, MIP131-143 h, and MIP149-164 m peptides showed the best interaction with the critical residues of TRAF2. Thus, these three peptides were used for generating peptidomimetics using pepMMsMIMIC, a peptidomimetics virtual screening tool. Around 600 peptidomimetics were identified & and retrieved for further screening by employing molecular docking tools and MD (Molecular Dynamics) simulations. Density Functional Theory (DFT) and ADMET predictions were applied to validate the screened peptidomimetics druggability. In the results, peptidomimic compounds MMs03918858 and MMs03927281 with binding energy values of -9.6 kcal/mol and - 9.1 kcal/mol respectively were screened as the best and are proposed for further pre-clinical studies.

Keywords:  ADMET; Alanine scanning; Atherosclerosis; CVD; Peptidomimetics

DOI:  https://doi.org/10.1007/s40203-023-00166-0
Neurobiol Dis. 2023 Oct 31. pii: S0969-9961(23)00358-3. [Epub ahead of print] 106342

A novel cell-permeable peptide prevents protein sumoylation and supports the mislocalization and aggregation of TDP-43.

R Marino, L Buccarello, K Hassanzadeh, K Akhtari, S Palaniappan, M Corbo, M Feligioni.

  SUMOylation is a post-translational modification (PTM) that exerts a regulatory role in different cellular processes, including protein localization, aggregation, and biological activities. It consists of the dynamic formation of covalent isopeptide bonds between a family member of the Small Ubiquitin Like Modifiers (SUMOs) and the target proteins. Interestingly, it is a cellular mechanism implicated in several neurodegenerative pathologies and potentially it could become a new therapeutic target; however, there are very few pharmacological tools to modulate the SUMOylation process. In this study, we have designed and tested the activity of a novel small cell-permeable peptide, COV-1, in a neuroblastoma cell line that specifically prevents protein SUMOylation. COV-1 inhibits UBC9-protein target interaction and efficiently decreases global SUMO-1ylation. Moreover, it can perturb RanGAP-1 perinuclear localization by inducing the downregulation of UBC9. In parallel, we found that COV-1 causes an increase in the ubiquitin degradation system up to its engulfment while enhancing the autophagic flux. Surprisingly, COV-1 modifies protein aggregation, and specifically it mislocalizes TDP-43 within cells, inducing its aggregation and co-localization with SUMO-1. These data suggest that COV-1 could be taken into future consideration as an interesting pharmacological tool to study the cellular cascade effects of SUMOylation prevention.

Keywords:  Aggregation; Autophagy; Cell-permeable peptides; Mislocalization; Protein target; RanGAP-1; SUMOylation; TDP43; UBC9; Ubiquitination

DOI:  https://doi.org/10.1016/j.nbd.2023.106342
Nano Lett. 2023 Nov 02.

Imaging Proteins Sensitive to Direct Fusions Using Transient Peptide-Peptide Interactions.

Zoe Gidden, Curran Oi, Emily J Johnston, Zuzanna Konieczna, Haresh Bhaskar, Lorena Mendive-Tapia, Fabio de Moliner, Susan J Rosser, Simon G J Mochrie, Marc Vendrell, Mathew H Horrocks, Lynne Regan.

  Fluorescence microscopy enables specific visualization of proteins in living cells and has played an important role in our understanding of the protein subcellular location and function. Some proteins, however, show altered localization or function when labeled using direct fusions to fluorescent proteins, making them difficult to study in live cells. Additionally, the resolution of fluorescence microscopy is limited to ∼200 nm, which is 2 orders of magnitude larger than the size of most proteins. To circumvent these challenges, we previously developed LIVE-PAINT, a live-cell super-resolution approach that takes advantage of short interacting peptides to transiently bind a fluorescent protein to the protein-of-interest. Here, we successfully use LIVE-PAINT to image yeast membrane proteins that do not tolerate the direct fusion of a fluorescent protein by using peptide tags as short as 5-residues. We also demonstrate that it is possible to resolve multiple proteins at the nanoscale concurrently using orthogonal peptide interaction pairs.

Keywords:  LIVE-PAINT; live-cell imaging; membrane protein; protein−protein interaction; super-resolution microscopy; yeast

DOI:  https://doi.org/10.1021/acs.nanolett.3c03780
J Neurovirol. 2023 Oct 29.

Disruption of blood-brain barrier: effects of HIV Tat on brain microvascular endothelial cells and tight junction proteins.

Yuqing Sun, Miaotian Cai, Ying Liang, Yulin Zhang.

  Although the widespread use of antiretroviral therapy (ART) has prolonged the life span of people living with HIV (PLWH), the incidence of HIV-associated neurocognitive disorders (HAND) in PLWH is also gradually increasing, seriously affecting the quality of life for PLWH. However, the pathogenesis of HAND has not been elucidated, which leaves HAND without effective treatment. HIV protein transactivator of transcription (Tat), as an important regulatory protein, is crucial in the pathogenesis of HAND, and its mechanism of HAND has received widespread attention. The blood-brain barrier (BBB) and its cellular component brain microvascular endothelial cells (BMVECs) play a necessary role in protecting the central nervous system (CNS), and their damage associated with Tat is a potential therapeutic target of HAND. In this review, we will study the Tat-mediated damage mechanism of the BBB and present multiple lines of evidence related to BMVEC damage caused by Tat.

Keywords:  Apoptosis; Blood–brain barrier; Brain microvascular endothelial cells; HIV Tat; HIV-associated neurocognitive disorders

DOI:  https://doi.org/10.1007/s13365-023-01179-3
Immunology. 2023 Oct 30.

PD-1 expression on tumour-infiltrating cells is a prognostic factor for relapsed or refractory diffuse large B-cell lymphoma.

Fumiya Wada, Kimimori Kamijo, Yoshimitsu Shimomura, Daisuke Yamashita, Shigeo Hara, Takayuki Ishikawa.

   BACKGROUND: The tumour microenvironment (TME), which is modulated after immune-chemotherapy, is involved in tumour growth and metastasis. Programmed cell death 1 (PD-1) expressed on tumour-infiltrating non-malignant cells plays an important role in the TME through the PD-1/programmed cell death ligand 1 (PD-L1) signalling pathway. However, its impact in patients with relapsed or refractory (R/R) diffuse large B-cell lymphoma (DLBCL) remains unclear.
METHODS: We conducted a retrospective study using tissue samples at relapse for patients with R/R DLBCL (n = 45) and evaluated the clinical impact of PD-1 expression on tumour-infiltrating non-malignant cells (microenvironmental PD-1, mPD-1). In addition, corresponding 27 samples at diagnosis were analysed to evaluate the changes in PD-1/PD-L1 expression in the TME after chemotherapy.
RESULTS: Patients with mPD-1+ DLBCL showed significantly better overall survival compared with patients with mPD-1- DLBCL (hazard ratio, 0.30, p = 0.03). Among patients with mPD-1- DLBCL, those positive for neoplastic or microenvironmental PD-L1 (nPD-L1+ or mPD-L1+ ) showed significantly worse outcomes. Microenvironmental PD-1 and PD-L1 expression has high correlation at relapse, although none was found at diagnosis.
CONCLUSION: We determined the clinical impact of microenvironmental PD-1 expression and its relationship with neoplastic or microenvironmental expression of PD-L1 in patients with R/R DLBCL. The expression of PD-1 and PD-L1 in the TME dramatically changes during the chemotherapy. Therefore, evaluating TME at relapse, not at diagnosis is useful to predict the outcomes of R/R DLBCL patients.

Keywords:  cancer; immunotherapy; lymphoid architecture; tumour immunology

DOI:  https://doi.org/10.1111/imm.13711
BMC Biol. 2023 Oct 31. 21(1): 238

TPpred-LE: therapeutic peptide function prediction based on label embedding.

Hongwu Lv, Ke Yan, Bin Liu.

   BACKGROUND: Therapeutic peptides play an essential role in human physiology, treatment paradigms and bio-pharmacy. Several computational methods have been developed to identify the functions of therapeutic peptides based on binary classification and multi-label classification. However, these methods fail to explicitly exploit the relationship information among different functions, preventing the further improvement of the prediction performance. Besides, with the development of peptide detection technology, peptide functions will be more comprehensively discovered. Therefore, it is necessary to explore computational methods for detecting therapeutic peptide functions with limited labeled data.
RESULTS: In this study, a novel method called TPpred-LE based on Transformer framework was proposed for predicting therapeutic peptide multiple functions, which can explicitly extract the function correlation information by using label embedding methodology and exploit the specificity information based on function-specific classifiers. Besides, we incorporated the multi-label classifier retraining approach (MCRT) into TPpred-LE to detect the new therapeutic functions with limited labeled data. Experimental results demonstrate that TPpred-LE outperforms the other state-of-the-art methods, and TPpred-LE with MCRT is robust for the limited labeled data.
CONCLUSIONS: In summary, TPpred-LE is a function-specific classifier for accurate therapeutic peptide function prediction, demonstrating the importance of the relationship information for therapeutic peptide function prediction. MCRT is a simple but effective strategy to detect functions with limited labeled data.

Keywords:  Multi-label classification; Multi-label classifier retrain; Relationship information; Therapeutic peptide prediction

DOI:  https://doi.org/10.1186/s12915-023-01740-w
J Med Chem. 2023 Oct 31.

Development of a 18F-Labeled Bicyclic Peptide Targeting EphA2 for Molecular Imaging of PSMA-Negative Prostate Cancer.

Qianqian Gan, Kai Cui, Qi Cao, Ning Zhang, Min-Fu Yang, Xing Yang.

Although PSMA PET/CT imaging has great potential for noninvasively detecting prostate cancer (PCa), limitations exist for patients with low PSMA expression, caused by androgen deprivation treatment or neuroendocrine differentiation. Analysis of The Cancer Genome Atlas Prostate Adenocarcinoma (TCGA-PRAD) data found that erythropoietin-producing hepatocellular receptor A2 (EphA2), a receptor overexpressed in most PCa could be a potential target for PSMA-negative PCa. A fluorescent ligand ETF and a radiolabeled ligand [18F]AlF-ETN derived from a EphA2-targeting bicyclic peptide were synthesized and investigated. ETF could selectively stain and visualize the EphA2-positive but PSMA-negative PC3 cells, in complementary to the PSMA-targeting probe. PET/CT imaging and biodistribution experiments demonstrated that [18F]AlF-ETN specifically accumulated in PC3 tumors with a high contrast (tumor-to-muscle ratio: 21.29 ± 6.55). In conclusion, we have demonstrated the potential for using EphA2 to detect PSMA-negative PCa and developed a radiolabeled ligand [18F]AlF-ETN to specifically image EphA2 expressing PCa with high contrast.

DOI: https://doi.org/10.1021/acs.jmedchem.3c01135
Nat Commun. 2023 Nov 01. 14(1): 6992

Recognition and reprogramming of E3 ubiquitin ligase surfaces by α-helical peptides.

Olena S Tokareva, Kunhua Li, Tara L Travaline, Ty M Thomson, Jean-Marie Swiecicki, Mahmoud Moussa, Jessica D Ramirez, Sean Litchman, Gregory L Verdine, John H McGee.

Molecules that induce novel interactions between proteins hold great promise for the study of biological systems and the development of therapeutics, but their discovery has been limited by the complexities of rationally designing interactions between three components, and because known binders to each protein are typically required to inform initial designs. Here, we report a general and rapid method for discovering α-helically constrained (Helicon) polypeptides that cooperatively induce the interaction between two target proteins without relying on previously known binders or an intrinsic affinity between the proteins. We show that Helicons are capable of binding every major class of E3 ubiquitin ligases, which are of great biological and therapeutic interest but remain largely intractable to targeting by small molecules. We then describe a phage-based screening method for discovering "trimerizer" Helicons, and apply it to reprogram E3s to cooperatively bind an enzyme (PPIA), a transcription factor (TEAD4), and a transcriptional coactivator (β-catenin).

DOI: https://doi.org/10.1038/s41467-023-42395-z
J Med Chem. 2023 Nov 03.

Covalent Peptide LSD1 Inhibitor Specifically Recognizes Cys360 in the Enzyme-Active Region.

Qinhong Luo, Yue Ma, Huiting Liang, Yuan Feng, Na Liu, Chenshan Lian, Lizhi Zhu, Yuxin Ye, Zhihong Liu, Zhanfeng Hou, Sijin Chen, Yaqi Wang, Chuan Dai, Chunli Song, Min Zhang, Zhipeng He, Yun Xing, Wanjin Zhong, Shuiming Li, Jianlong Wu, Fei Lu, Feng Yin, Zigang Li.

Lysine-specific demethylase 1 (LSD1) is a promising therapeutic target, especially in cancer treatment. Despite several LSD1 inhibitors being discovered for the cofactor pocket, none are FDA-approved. We aimed to develop stabilized peptides for irreversible LSD1 binding, focusing on unique cysteine residue Cys360 in LSD1 and SNAIL1. We created LSD1 C360-targeting peptides, like cyclic peptide S9-CMC1, using our Cysteine-Methionine cyclization strategy. S9-CMC1 effectively inhibited LSD1 at the protein level, as confirmed by MS analysis showing covalent bonding to Cys360. In cells, S9-CMC1 inhibited LSD1 activity, increasing H3K4me1 and H3K4me2 levels, leading to G1 cell cycle arrest and apoptosis and inhibiting cell proliferation. Remarkably, S9-CMC1 showed therapeutic potential in A549 xenograft animal models, regulating LSD1 activity and significantly inhibiting tumor growth with minimal organ damage. These findings suggest LSD1 C360 as a promising site for covalent LSD1 inhibitors' development.

DOI: https://doi.org/10.1021/acs.jmedchem.3c01549
Curr Protein Pept Sci. 2023 Oct 25.

Bioconjugation Techniques for Enhancing Stability and Targeting Efficiency of Protein and Peptide Therapeutics.

Tanuja Bisht, Anupriya Adhikari, Shivanand Patil, Shivang Dhoundiyal.

  Bioconjugation techniques have emerged as powerful tools for enhancing the stability and targeting efficiency of protein and peptide therapeutics. This review provides a comprehensive analysis of the various bioconjugation strategies employed in the field. The introduction highlights the significance of bioconjugation techniques in addressing stability and targeting challenges associated with protein and peptide-based drugs. Chemical and enzymatic bioconjugation methods are discussed, along with crosslinking strategies for covalent attachment and site-specific conjugation approaches. The role of bioconjugation in improving stability profiles is explored, showcasing case studies that demonstrate successful stability enhancement. Furthermore, bioconjugation techniques for ligand attachment and targeting are presented, accompanied by examples of targeted protein and peptide therapeutics. The review also covers bioconjugation approaches for prolonging circulation and controlled release, focusing on strategies to extend half-life, reduce clearance, and design-controlled release systems. Analytical characterization techniques for bioconjugates, including the evaluation of conjugation efficiency, stability, and assessment of biological activity and targeting efficiency, are thoroughly examined. In vivo considerations and clinical applications of bioconjugated protein and peptide therapeutics, including pharmacokinetic and pharmacodynamic considerations, as well as preclinical and clinical developments, are discussed. Finally, the review concludes with an overview of future perspectives, emphasizing the potential for novel conjugation methods and advanced targeting strategies to further enhance the stability and targeting efficiency of protein and peptide therapeutics.

Keywords:  Bioconjugation; crosslinking; peptide; protein; site-specific; stability.

DOI:  https://doi.org/10.2174/0113892037268777231013154850
Chem Biodivers. 2023 Nov 01. e202300399

Triptolide Reduces MDA-MB-231 Cell Metastasis by Attenuating Epithelial-Mesenchymal Transition through the ROCK/PTEN/Akt Axis.

Qinhang Wu, Xuejiao Leng, Xuelin Ma, Qixuan Xu, Ruijun Ni, Yang Pan, Zhuolin Jin, Muhammad Kalim.

  Triple-negative breast cancer (TNBC) is a highly heterogeneous and invasive subtype of breast cancer. The prognosis of TNBC is poor because of its high distant metastasis rate. Triptolide is a type of diterpene trioxide natural compound with potential anti-tumor activities. This study explored the metastatic inhibitory effect of triptolide on MDA-MB-231 cells and its underlying mechanism. Triptolide suppressed cell proliferation and induced cell apoptosis in a time- and dose-dependent manner. Low doses of triptolide (0-8 nM) reduced the migration and invasion capabilities of MDA-MB-231 cells. Triptolide decreased ROCK1, p-Akt, N-cadherin, vimentin and MMP-9 expressions, but increased PTEN and E-cadherin expressions on protein and mRNA levels. Furthermore, the down-regulation of ROCK1 expression in MDA-MB-231 cells after being treated by triptolide could be rescued by ROCK1 specific inhibitor Y27632. Molecular docking showed that triptolide and Y27632 shared the same active center of ROCK1 protein. This article's findings taken together showed that ROCK1 is the primary target of triptolide, which can cause cell apoptosis and inhibit the epithelial-mesenchymal transition of MDA-MB-231 cells.

Keywords:  Apoptosis; MDA-MB-231 cell; ROCK1; metastasis; natural compound

DOI:  https://doi.org/10.1002/cbdv.202300399
Cell Rep Methods. 2023 Oct 26. pii: S2667-2375(23)00288-6. [Epub ahead of print] 100624

B2LiVe, a label-free 1D-NMR method to quantify the binding of amphitropic peptides or proteins to membrane vesicles.

Mirko Sadi, Nicolas Carvalho, Corentin Léger, Bruno Vitorge, Daniel Ladant, J Iñaki Guijarro, Alexandre Chenal.

  Amphitropic proteins and peptides reversibly partition from solution to membrane, a key process that regulates their functions. Experimental approaches classically used to measure protein partitioning into lipid bilayers, such as fluorescence and circular dichroism, are hardly usable when the peptides or proteins do not exhibit significant polarity and/or conformational changes upon membrane binding. Here, we describe binding to lipid vesicles (B2LiVe), a simple, robust, and widely applicable nuclear magnetic resonance (NMR) method to determine the solution-to-membrane partitioning of unlabeled proteins or peptides. B2LiVe relies on previously described proton 1D-NMR fast-pulsing techniques. Membrane partitioning induces a large line broadening, leading to a loss of protein signals; therefore, the decrease of the NMR signal directly measures the fraction of membrane-bound protein. The method uses low polypeptide concentrations and has been validated on several membrane-interacting polypeptides, ranging from 3 to 54 kDa, with membrane vesicles of different sizes and various lipid compositions.

Keywords:  CP: Cell biology; CP: Molecular biology; affinity; amphitropic proteins; nuclear magnetic resonance; partition coefficient; peptides; protein membrane binding; proton 1D-NMR fast-pulsing techniques; selective adiabatic pulses; solution to membrane partitioning

DOI:  https://doi.org/10.1016/j.crmeth.2023.100624