bims-cepepe 2023-10-29 papers

bims-cepepe

Biomed News

on Cell-penetrating peptides

Issue of 2023–10–29
twenty-six papers selected by
Henry Lamb, Queensland University of Technology

Macrocyclic Peptides Closed by a Thioether-Bipyridyl Unit That Grants Cell Membrane Permeability.
Topoisomeric Membrane-Active Peptides: A Review of the Last Two Decades.
Cyclic Peptides for Drug Development.
SaLT&PepPr is an interface-predicting language model for designing peptide-guided protein degraders.
Amphipathic Cell-Penetrating Peptide-Aided Delivery of Cas9 RNP for In Vitro Gene Editing and Correction.
PepMLM: Target Sequence-Conditioned Generation of Peptide Binders via Masked Language Modeling.
A cell-penetrating peptide derived from SARS-CoV-2 protein Orf9b allosterically inhibits MARK4 activity and mitigates tau toxicity.
Improve BBB Penetration and Cytotoxicity of Palbociclib in U87-MG Glioblastoma Cells Delivered by Dual Peptide Functionalized Nanoparticles.
Chemokine-derived oncolytic peptide induces immunogenic cancer cell death and significantly suppresses tumor growth.
Validation of a New Methodology to Create Oral Drugs beyond the Rule of 5 for Intracellular Tough Targets.
Develop a PD-1-blockade peptide to reinvigorate T-cell activity and inhibit tumor progress.
Practical N-to-C peptide synthesis with minimal protecting groups.
Advances in biosynthesis of peptide drugs: Technology and industrialization.
A novel genetically-encoded bicyclic peptide inhibitor of human urokinase-type plasminogen activator with better cross-reactivity toward the murine orthologue.
FDA approves C5-blocking macrocyclic peptide for autoimmune disease.
An Induced Pluripotent Stem Cell-Derived Human Blood-Brain Barrier (BBB) Model to Test the Crossing by Adeno-Associated Virus (AAV) Vectors and Antisense Oligonucleotides.
Peptides from Marine-Derived Fungi: Chemistry and Biological Activities.
The Membrane-Mediated Interaction of Liquid-Ordered Lipid Domains in the Presence of Amphipathic Peptides.
An amino-domino model described by a cross-peptide-bond Ramachandran plot defines amino acid pairs as local structural units.
Synthesis of DNA-Encoded Macrocyclic Peptides via Nitrile-Aminothiol Click Reaction.
Peptide binder design with inverse folding and protein structure prediction.
Discovery of an Insect Neuroactive Helix Ring Peptide from Ant Venom.
Substrates and Cyclic Peptide Inhibitors of the Oligonucleotide Activated Sirtuin 7.
Tandem Mass Spectrometry de novo Sequencing of the Skin Defense Peptides of the Central Slovenian Agile Frog Rana dalmatina.
Compromised Blood-Brain Barrier Junctions Enhance Melanoma Cell Intercalation and Extravasation.
Long Prehensile Protrusions Can Facilitate Cancer Cell Invasion through the Basement Membrane.

ACS Bio Med Chem Au. 2023 Oct 18. 3(5): 429-437

Macrocyclic Peptides Closed by a Thioether-Bipyridyl Unit That Grants Cell Membrane Permeability.

Hongxue Chen, Takayuki Katoh, Hiroaki Suga.

Membrane permeability is an important factor that determines the virtue of peptides targeting intracellular molecules. By introducing a membrane penetration motif, some peptides exhibit better membrane permeabilities. Previous choices for such motifs have usually been polycationic sequences, but their protease vulnerabilities and modest endosome escapability remain challenging. Here, we report a strategy for macrocyclization of peptides closed by a hydrophobic bipyridyl (BPy) unit, which grants an improvement of their membrane permeability and proteolytic stability compared with the conventional polycationic peptides. We chemically prepared model macrocyclic peptides closed by a thioether-BPy unit and determined their cell membrane permeability, giving 200 nM CP50 (an indicative value of membrane permeability), which is 40-fold better than that of the ordinary thioether macrocycle consisting of the same sequence composition. To discover potent target binders consisting of the BPy unit, we reprogrammed the initiator with chloromethyl-BPy (ClMeBPy) for the peptide library synthesis with a downstream Cys residue(s) and executed RaPID (Random nonstandard Peptide Integrated Discovery) against the bromodomains of BRD4. One of the obtained sequences exhibited a single-digit nanomolar dissociation constant against BRD4 in vitro and showed approximately 2-fold and 10-fold better membrane permeability than positive controls, R9 and Tat peptides, respectively. Moreover, we observed an intracellular activity of the BPy macrocycle tagged with a proteasome target peptide motif (RRRG), resulting in modest but detectable degradation of BRD4. The present demonstration indicates that the combination of the RaPID system with an appropriate hydrophobic unit, such as BPy, would provide a potential approach for devising cell penetrating macrocycles targeting various intracellular proteins.

DOI: https://doi.org/10.1021/acsbiomedchemau.3c00027
Pharmaceutics. 2023 Oct 12. pii: 2451. [Epub ahead of print]15(10):

Topoisomeric Membrane-Active Peptides: A Review of the Last Two Decades.

Adam Carrera-Aubesart, Maria Gallo, Sira Defaus, Toni Todorovski, David Andreu.

  In recent decades, bioactive peptides have been gaining recognition in various biomedical areas, such as intracellular drug delivery (cell-penetrating peptides, CPPs) or anti-infective action (antimicrobial peptides, AMPs), closely associated to their distinct mode of interaction with biological membranes. Exploiting the interaction of membrane-active peptides with diverse targets (healthy, tumoral, bacterial or parasitic cell membranes) is opening encouraging prospects for peptides in therapeutics. However, ordinary peptides formed by L-amino acids are easily decomposed by proteases in biological fluids. One way to sidestep this limitation is to use topoisomers, namely versions of the peptide made up of D-amino acids in either canonic (enantio) or inverted (retroenantio) sequence. Rearranging peptide sequences in this fashion provides a certain degree of native structure mimicry that, in appropriate contexts, may deliver desirable biological activity while avoiding protease degradation. In this review, we will focus on recent accounts of membrane-active topoisomeric peptides with therapeutic applications as CPP drug delivery vectors, or as antimicrobial and anticancer candidates. We will also discuss the most common modes of interaction of these peptides with their membrane targets.

Keywords:  anticancer peptides; antimicrobial peptides; cell-penetrating peptides; enantio; membrane-active peptides; retro; retroenantio; topoisomery

DOI:  https://doi.org/10.3390/pharmaceutics15102451
Angew Chem Int Ed Engl. 2023 Oct 23. e202308251

Cyclic Peptides for Drug Development.

Xinjian Ji, Alexander L Nielsen, Christian Heinis.

  Cyclic peptides are fascinating molecules abundantly found in nature and exploited as molecular format for drug development as well as other applications, ranging from research tools to food additives. Advances in peptide technologies made over many years through improved methods for synthesis and drug development have resulted in a steady stream of new drugs, with an average of around one cyclic peptide drug approved per year. Powerful technologies for screening random peptide libraries, and de novo generating ligands, have enabled the development of cyclic peptide drugs independent of naturally derived molecules and now offer virtually unlimited development opportunities. In this review, we feature therapeutically relevant cyclic peptides derived from nature and discuss the unique properties of cyclic peptides, the enormous technological advances in peptide ligand development in recent years, and current challenges and opportunities for developing cyclic peptides that address unmet medical needs.

Keywords:  Cyclic Peptide; Macrocycle; Peptide; Peptide Drug; Peptide Therapeutics

DOI:  https://doi.org/10.1002/anie.202308251
Commun Biol. 2023 Oct 24. 6(1): 1081

SaLT&PepPr is an interface-predicting language model for designing peptide-guided protein degraders.

Garyk Brixi, Tianzheng Ye, Lauren Hong, Tian Wang, Connor Monticello, Natalia Lopez-Barbosa, Sophia Vincoff, Vivian Yudistyra, Lin Zhao, Elena Haarer, Tianlai Chen, Sarah Pertsemlidis, Kalyan Palepu, Suhaas Bhat, Jayani Christopher, Xinning Li, Tong Liu, Sue Zhang, Lillian Petersen, Matthew P DeLisa, Pranam Chatterjee.

Protein-protein interactions (PPIs) are critical for biological processes and predicting the sites of these interactions is useful for both computational and experimental applications. We present a Structure-agnostic Language Transformer and Peptide Prioritization (SaLT&PepPr) pipeline to predict interaction interfaces from a protein sequence alone for the subsequent generation of peptidic binding motifs. Our model fine-tunes the ESM-2 protein language model (pLM) with a per-position prediction task to identify PPI sites using data from the PDB, and prioritizes motifs which are most likely to be involved within inter-chain binding. By only using amino acid sequence as input, our model is competitive with structural homology-based methods, but exhibits reduced performance compared with deep learning models that input both structural and sequence features. Inspired by our previous results using co-crystals to engineer target-binding "guide" peptides, we curate PPI databases to identify partners for subsequent peptide derivation. Fusing guide peptides to an E3 ubiquitin ligase domain, we demonstrate degradation of endogenous β-catenin, 4E-BP2, and TRIM8, and highlight the nanomolar binding affinity, low off-targeting propensity, and function-altering capability of our best-performing degraders in cancer cells. In total, our study suggests that prioritizing binders from natural interactions via pLMs can enable programmable protein targeting and modulation.

DOI: https://doi.org/10.1038/s42003-023-05464-z
Pharmaceutics. 2023 Oct 20. pii: 2500. [Epub ahead of print]15(10):

Amphipathic Cell-Penetrating Peptide-Aided Delivery of Cas9 RNP for In Vitro Gene Editing and Correction.

Mert Öktem, Enrico Mastrobattista, Olivier G de Jong.

  The therapeutic potential of the CRISPR-Cas9 gene editing system in treating numerous genetic disorders is immense. To fully realize this potential, it is crucial to achieve safe and efficient delivery of CRISPR-Cas9 components into the nuclei of target cells. In this study, we investigated the applicability of the amphipathic cell-penetrating peptide LAH5, previously employed for DNA delivery, in the intracellular delivery of spCas9:sgRNA ribonucleoprotein (RNP) and the RNP/single-stranded homology-directed repair (HDR) template. Our findings reveal that the LAH5 peptide effectively formed nanocomplexes with both RNP and RNP/HDR cargo, and these nanocomplexes demonstrated successful cellular uptake and cargo delivery. The loading of all RNP/HDR components into LAH5 nanocomplexes was confirmed using an electrophoretic mobility shift assay. Functional screening of various ratios of peptide/RNP nanocomplexes was performed on fluorescent reporter cell lines to assess gene editing and HDR-mediated gene correction. Moreover, targeted gene editing of the CCR5 gene was successfully demonstrated across diverse cell lines. This LAH5-based delivery strategy represents a significant advancement toward the development of therapeutic delivery systems for CRISPR-Cas-based genetic engineering in in vitro and ex vivo applications.

Keywords:  CRISPR-Cas9; HDR; LAH5; RNP; cell-penetrating peptide (CPP); delivery

DOI:  https://doi.org/10.3390/pharmaceutics15102500
ArXiv. 2023 Oct 05. pii: arXiv:2310.03842v1. [Epub ahead of print]

PepMLM: Target Sequence-Conditioned Generation of Peptide Binders via Masked Language Modeling.

Tianlai Chen, Sarah Pertsemlidis, Venkata Srikar Kavirayuni, Pranay Vure, Rishab Pulugurta, Ashley Hsu, Sophia Vincoff, Vivian Yudistyra, Lauren Hong, Tian Wang, Elena Haarer, Lin Zhao, Pranam Chatterjee.

Target proteins that lack accessible binding pockets and conformational stability have posed increasing challenges for drug development. Induced proximity strategies, such as PROTACs and molecular glues, have thus gained attention as pharmacological alternatives, but still require small molecule docking at binding pockets for targeted protein degradation (TPD). The computational design of protein-based binders presents unique opportunities to access "undruggable" targets, but have often relied on stable 3D structures or predictions for effective binder generation. Recently, we have leveraged the expressive latent spaces of protein language models (pLMs) for the prioritization of peptide binders from sequence alone, which we have then fused to E3 ubiquitin ligase domains, creating a CRISPR-analogous TPD system for target proteins. However, our methods rely on training discriminator models for ranking heuristically or unconditionally-derived "guide" peptides for their target binding capability. In this work, we introduce PepMLM, a purely target sequence-conditioned de novo generator of linear peptide binders. By employing a novel masking strategy that uniquely positions cognate peptide sequences at the terminus of target protein sequences, PepMLM tasks the state-of-the-art ESM-2 pLM to fully reconstruct the binder region, achieving low perplexities matching or improving upon previously-validated peptide-protein sequence pairs. After successful in silico benchmarking with AlphaFold-Multimer, we experimentally verify PepMLM's efficacy via fusion of model-derived peptides to E3 ubiquitin ligase domains, demonstrating endogenous degradation of target substrates in cellular models. In total, PepMLM enables the generative design of candidate binders to any target protein, without the requirement of target structure, empowering downstream programmable proteome editing applications.
Neurobiol Dis. 2023 Oct 24. pii: S0969-9961(23)00350-9. [Epub ahead of print] 106334

A cell-penetrating peptide derived from SARS-CoV-2 protein Orf9b allosterically inhibits MARK4 activity and mitigates tau toxicity.

Toshiya Oba, Daiki Homma, Sophia Jobien M Limlingan, Aoi Fukuchi, Akiko Asada, Taro Saito, Kanae Ando.

  Abnormal activation of microtubule affinity-regulating kinase 4 (MARK4) and its phosphorylation of the microtubule-associated protein tau are believed to play a role in the pathogenesis of Alzheimer's disease, and MARK4 inhibition can be a strategy to develop disease-modifying therapy. Here we report the development of a membrane-permeable peptide that inhibits MARK4 activity in an allosteric manner. The SARS-CoV-2-derived protein Orf9b inhibited MARK4-mediated tau phosphorylation in primary neurons and Drosophila. Orf9b inhibited MARK4 activity in an allosteric manner and did not inhibit the activity of MARK2, which is another MARK family member and is closely related to MARK4. Co-expression of Orf9b in the fly retina expressing human tau and MARK4 suppressed phosphorylation of tau at the microtubule-binding repeats and tau-induced neurodegeneration. We identified the minimal sequence of Orf9b required to suppress MARK4 activity and fused it to a cell-permeable sequence (TAT-Orf9b10-18_78-95). Extracellular supplementation of TAT-Orf9b10-18_78-95 inhibited MARK4 activity in primary neurons, and feeding TAT-Orf9b10-18_78-95 to a fly model of tauopathy lowered phospho-tau levels and suppressed neurodegeneration. These results suggest that TAT-Orf9b10-18_78-95 is a unique class of MARK4 inhibitor and can be used to modify tau toxicity.

Keywords:  Allosteric; Cell-membrane permeable peptides; Drosophila model of disease; Microtubule affinity-regulating kinase 4; Neurodegeneration; Peptide inhibitor for protein kinases; Tau

DOI:  https://doi.org/10.1016/j.nbd.2023.106334
Pharmaceutics. 2023 Oct 06. pii: 2429. [Epub ahead of print]15(10):

Improve BBB Penetration and Cytotoxicity of Palbociclib in U87-MG Glioblastoma Cells Delivered by Dual Peptide Functionalized Nanoparticles.

Yu-Chen Lo, Wen-Jen Lin.

  Palbociclib (PBC) is an FDA-approved CDK4/6 inhibitor used for breast cancer treatment. PBC has been demonstrated its ability to suppress the proliferation of glioma cells by inducing cell cycle arrest. However, the efflux transporters on the blood-brain barrier (BBB) restricts the delivery of PBC to the brain. The nano-delivery strategy with BBB-penetrating and glioma-targeting abilities was designed. Poly(lactide-co-glycolide)-poly(ethylene glycol) (PLGA-PEG) was functionalized with the potential peptide, T7 targeting peptide and/or R9 penetrating peptide, to prepare PBC-loaded nanoparticles (PBC@NPs). The size of PBC@NPs was in the range of 168.4 ± 4.3-185.8 ± 4.4 nm (PDI < 0.2), and the zeta potential ranged from -17.8 ± 1.4 mV to -14.3 ± 1.0 mV dependent of conjugated peptide. The transport of PBC@NPs across the bEnd.3 cell model was in the order of dual-peptide modified NPs > T7-peptide modified NPs > peptide-free NPs > free PBC, indicating facilitated delivery of PBC by NPs, particularly the T7/R9 dual-peptide modified NPs. Moreover, PBC@NPs significantly enhanced U87-MG glioma cell apoptosis by 2.3-6.5 folds relative to PBC, where the dual-peptide modified NPs was the most effective one. In conclusion, the PBC loaded dual-peptide functionalized NPs improved cellular uptake in bEnd.3 cells followed by targeting to U87-MG glioma cells, leading to effective cytotoxicity and promoting cell death.

Keywords:  dual peptide; glioma cells; nanoparticles; palbociclib

DOI:  https://doi.org/10.3390/pharmaceutics15102429
Res Sq. 2023 Oct 03. pii: rs.3.rs-3335225. [Epub ahead of print]

Chemokine-derived oncolytic peptide induces immunogenic cancer cell death and significantly suppresses tumor growth.

Natsuki Furukawa, Wendy Yang, Alex Chao, Akash Patil, Adam Mirando, Niranjan Pandey, Aleksander Popel.

Chemokinostatin-1 (CKS1) is a 24-mer peptide originally discovered as an anti-angiogenic peptide derived from the CXCL1 chemokine. Here, we demonstrate that CKS1 acts not only as an anti-angiogenic peptide but also as an oncolytic peptide due to its structural and physical properties. CKS1 induced both necrotic and apoptotic cell death specifically in cancer cells while showing minimal toxicity in non-cancerous cells. Mechanistically, CKS1 disrupted the cell membrane of cancer cells quickly after treatment and activated the apoptotic pathway at later time points. Furthermore, immunogenic molecules were released from CKS1 treated cells, indicating that CKS1 induces immunogenic cell death. CKS1 effectively suppressed tumor growth in vivo . Collectively, these data demonstrate that CKS1 is a unique peptide that functions both as an anti-angiogenic peptide and as an oncolytic peptide and has a therapeutic potential to treat cancer.

DOI: https://doi.org/10.21203/rs.3.rs-3335225/v1
J Am Chem Soc. 2023 Oct 24.

Validation of a New Methodology to Create Oral Drugs beyond the Rule of 5 for Intracellular Tough Targets.

Atsushi Ohta, Mikimasa Tanada, Shojiro Shinohara, Yuya Morita, Kazuhiko Nakano, Yusuke Yamagishi, Ryusuke Takano, Shiori Kariyuki, Takeo Iida, Atsushi Matsuo, Kazuhisa Ozeki, Takashi Emura, Yuuji Sakurai, Koji Takano, Atsuko Higashida, Miki Kojima, Terushige Muraoka, Ryuuichi Takeyama, Tatsuya Kato, Kaori Kimura, Kotaro Ogawa, Kazuhiro Ohara, Shota Tanaka, Yasufumi Kikuchi, Nozomi Hisada, Ryuji Hayashi, Yoshikazu Nishimura, Kenichi Nomura, Tatsuhiko Tachibana, Machiko Irie, Hatsuo Kawada, Takuya Torizawa, Naoaki Murao, Tomoya Kotake, Masahiko Tanaka, Shiho Ishikawa, Taiji Miyake, Minoru Tamiya, Masako Arai, Aya Chiyoda, Sho Akai, Hitoshi Sase, Shino Kuramoto, Toshiya Ito, Takuya Shiraishi, Tetsuo Kojima, Hitoshi Iikura.

Establishing a technological platform for creating clinical compounds inhibiting intracellular protein-protein interactions (PPIs) can open the door to many valuable drugs. Although small molecules and antibodies are mainstream modalities, they are not suitable for a target protein that lacks a deep cavity for a small molecule to bind or a protein found in intracellular space out of an antibody's reach. One possible approach to access these targets is to utilize so-called middle-size cyclic peptides (defined here as those with a molecular weight of 1000-2000 g/mol). In this study, we validated a new methodology to create oral drugs beyond the rule of 5 for intracellular tough targets by elucidating structural features and physicochemical properties for drug-like cyclic peptides and developing library technologies to afford highly N-alkylated cyclic peptide hits. We discovered a KRAS inhibitory clinical compound (LUNA18) as the first example of our platform technology.

DOI: https://doi.org/10.1021/jacs.3c07145
Eur J Pharmacol. 2023 Oct 20. pii: S0014-2999(23)00658-1. [Epub ahead of print] 176144

Develop a PD-1-blockade peptide to reinvigorate T-cell activity and inhibit tumor progress.

Yingzhou Qin, Xiangzhou Meng, Lin Li, Cuijuan Liu, Fan Gao, Xin Yuan, Ying Huang, Yimin Zhu.

  Immune checkpoint inhibitors, particularly monoclonal antibodies blocking the programmed cell death 1 (PD-1)/programmed cell death ligand-1 (PD-L1) pathway, have been successfully utilized in the clinic. However, certain drawbacks associated with antibodies, such as high immunogenicity and poor tissue penetration, need to be addressed for their broader clinical application. Peptides, as low molecular weight alternatives, have garnered increasing interest in this field. In this study, we employed bacterial surface display technology to identify a PD-1-binding peptide, PBP. The PBP peptide exhibited moderate affinity for human PD-1 (hPD-1) and displayed cross-reactivity with mouse PD-1 (mPD-1). Molecular docking analysis revealed that the interaction residues of the PBP peptide with PD-1 played crucial roles in the formation of the PD-1/PD-L1 complex. A competing binding assay demonstrated that the peptide could interfere the interaction of PD-1 and PD-L1. Moreover, in vitro experiments showed that the PBP peptide could reinvigorate T cells inhibited by PD-L1. In an in vivo mouse model of CT26, the PBP peptide effectively suppressed tumor growth by enhancing T cell function. In conclusion, our results suggest that the PBP peptide exerts an anti-tumor effect by impeding the interplay between PD-1 and PD-L1, highlighting its potential as an alternative for tumor immunotherapy.

Keywords:  Bacterial surface display; Checkpoint inhibitor; PD-1; PD-L1; Peptide; T cell

DOI:  https://doi.org/10.1016/j.ejphar.2023.176144
Commun Chem. 2023 Oct 26. 6(1): 231

Practical N-to-C peptide synthesis with minimal protecting groups.

Toshifumi Tatsumi, Koki Sasamoto, Takuya Matsumoto, Ryo Hirano, Kazuki Oikawa, Masato Nakano, Masaru Yoshida, Kounosuke Oisaki, Motomu Kanai.

Accessible drug modalities have continued to increase in number in recent years. Peptides play a central role as pharmaceuticals and biomaterials in these new drug modalities. Although traditional peptide synthesis using chain-elongation from C- to N-terminus is reliable, it produces large quantities of chemical waste derived from protecting groups and condensation reagents, which place a heavy burden on the environment. Here we report an alternative N-to-C elongation strategy utilizing catalytic peptide thioacid formation and oxidative peptide bond formation with main chain-unprotected amino acids under aerobic conditions. This method is applicable to both iterative peptide couplings and convergent fragment couplings without requiring elaborate condensation reagents and protecting group manipulations. A recyclable N-hydroxy pyridone additive effectively suppresses epimerization at the elongating chain. We demonstrate the practicality of this method by showcasing a straightforward synthesis of the nonapeptide DSIP. This method further opens the door to clean and atom-efficient peptide synthesis.

DOI: https://doi.org/10.1038/s42004-023-01030-0
Biotechnol J. 2023 Oct 26. e2300256

Advances in biosynthesis of peptide drugs: Technology and industrialization.

Jing Wang, Long Chen, Song Qin, Mingyuan Xie, Shi-Zhong Luo, Wenjun Li.

  Peptide drugs are developed from endogenous or synthetic peptides with specific biological activities. They have advantages of strong target specificity, high efficacy and low toxicity, thus showing great promise in the treatment of many diseases such as cancer, infections and diabetes. Although an increasing number of peptide drugs have entered market in recent years, the preparation of peptide drug substances is yet a bottleneck problem for their industrial production. Comparing to the chemical synthesis method, peptide biosynthesis has advantages of simple synthesis, low cost, and low contamination. Therefore, the biosynthesis technology of peptide drugs has been widely used for manufacturing. Herein, we reviewed the development of peptide drugs and recent advances in peptide biosynthesis technology, in order to shed a light to the prospect of industrial production of peptide drugs based on biosynthesis technology. This article is protected by copyright. All rights reserved.

Keywords:  biosynthesis technology; industrialization; peptide drugs; peptide synthesis

DOI:  https://doi.org/10.1002/biot.202300256
Bioorg Med Chem. 2023 Oct 12. pii: S0968-0896(23)00347-4. [Epub ahead of print]95 117499

A novel genetically-encoded bicyclic peptide inhibitor of human urokinase-type plasminogen activator with better cross-reactivity toward the murine orthologue.

Ylenia Mazzocato, Stefano Perin, Julia Morales-Sanfrutos, Zhanna Romanyuk, Stefano Pluda, Laura Acquasaliente, Giuseppe Borsato, Vincenzo De Filippis, Alessandro Scarso, Alessandro Angelini.

  The inhibition of human urokinase-type plasminogen activator (huPA), a serine protease that plays an important role in pericellular proteolysis, is a promising strategy to decrease the invasive and metastatic activity of tumour cells. However, the generation of selective small molecule huPA inhibitors has proven to be challenging due to the high structural similarity of huPA to other paralogue serine proteases. Efforts to generate more specific therapies have led to the development of cyclic peptide-based inhibitors with much higher selectivity against huPA. While this latter property is desired, the sparing of the orthologue murine poses difficulties for the testing of the inhibitor in preclinical mouse model. In this work, we have applied a Darwinian evolution-based approach to identify phage-encoded bicyclic peptide inhibitors of huPA with better cross-reactivity towards murine uPA (muPA). The best selected bicyclic peptide (UK132) inhibited huPA and muPA with Ki values of 0.33 and 12.58 µM, respectively. The inhibition appears to be specific for uPA, as UK132 only weakly inhibits a panel of structurally similar serine proteases. Removal or substitution of the second loop with one not evolved in vitro led to monocyclic and bicyclic peptide analogues with lower potency than UK132. Moreover, swapping of 1,3,5-tris-(bromomethyl)-benzene with different small molecules not used in the phage selection, resulted in an 80-fold reduction of potency, revealing the important structural role of the branched cyclization linker. Further substitution of an arginine in UK132 to a lysine resulted in a bicyclic peptide UK140 with enhanced inhibitory potency against both huPA (Ki = 0.20 µM) and murine orthologue (Ki = 2.79 µM). By combining good specificity, nanomolar affinity and a low molecular mass, the bicyclic peptide inhibitor developed in this work may provide a novel human and murine cross-reactive lead for the development of a potent and selective anti-metastatic therapy.

Keywords:  Affinity maturation; Cyclic peptide; Directed evolution; Macrocycle; Phage display; Urokinase-type plasminogen activator; uPA

DOI:  https://doi.org/10.1016/j.bmc.2023.117499
Nat Rev Drug Discov. 2023 Oct 25.

FDA approves C5-blocking macrocyclic peptide for autoimmune disease.

Asher Mullard.

DOI: https://doi.org/10.1038/d41573-023-00171-0
Biomedicines. 2023 Oct 04. pii: 2700. [Epub ahead of print]11(10):

An Induced Pluripotent Stem Cell-Derived Human Blood-Brain Barrier (BBB) Model to Test the Crossing by Adeno-Associated Virus (AAV) Vectors and Antisense Oligonucleotides.

Jamuna Selvakumaran, Simona Ursu, Melissa Bowerman, Ngoc Lu-Nguyen, Matthew J Wood, Alberto Malerba, Rafael J Yáñez-Muñoz.

  The blood-brain barrier (BBB) is the specialised microvasculature system that shields the central nervous system (CNS) from potentially toxic agents. Attempts to develop therapeutic agents targeting the CNS have been hindered by the lack of predictive models of BBB crossing. In vitro models mimicking the human BBB are of great interest, and advances in induced pluripotent stem cell (iPSC) technologies and the availability of reproducible differentiation protocols have facilitated progress. In this study, we present the efficient differentiation of three different wild-type iPSC lines into brain microvascular endothelial cells (BMECs). Once differentiated, cells displayed several features of BMECs and exhibited significant barrier tightness as measured by trans-endothelial electrical resistance (TEER), ranging from 1500 to >6000 Ωcm2. To assess the functionality of our BBB models, we analysed the crossing efficiency of adeno-associated virus (AAV) vectors and peptide-conjugated antisense oligonucleotides, both currently used in genetic approaches for the treatment of rare diseases. We demonstrated superior barrier crossing by AAV serotype 9 compared to serotype 8, and no crossing by a cell-penetrating peptide-conjugated antisense oligonucleotide. In conclusion, our study shows that iPSC-based models of the human BBB display robust phenotypes and could be used to screen drugs for CNS penetration in culture.

Keywords:  adeno-associated virus vectors; antisense oligonucleotides; brain microvascular endothelial cells; gene therapy; human blood–brain barrier; induced pluripotent stem cells; phosphorodiamidate morpholino oligomers; spinal muscular atrophy; trans-endothelial electrical resistance

DOI:  https://doi.org/10.3390/biomedicines11102700
Mar Drugs. 2023 Sep 26. pii: 510. [Epub ahead of print]21(10):

Peptides from Marine-Derived Fungi: Chemistry and Biological Activities.

Salar Hafez Ghoran, Fatemeh Taktaz, Emília Sousa, Carla Fernandes, Anake Kijjoa.

  Marine natural products are well-recognized as potential resources to fill the pipeline of drug leads to enter the pharmaceutical industry. In this circumstance, marine-derived fungi are one of the unique sources of bioactive secondary metabolites due to their capacity to produce diverse polyketides and peptides with unique structures and diverse biological activities. The present review covers the peptides from marine-derived fungi reported from the literature published from January 1991 to June 2023, and various scientific databases, including Elsevier, ACS publications, Taylor and Francis, Wiley Online Library, MDPI, Springer, Thieme, Bentham, ProQuest, and the Marine Pharmacology website, are used for a literature search. This review focuses on chemical characteristics, sources, and biological and pharmacological activities of 366 marine fungal peptides belonging to various classes, such as linear, cyclic, and depsipeptides. Among 30 marine-derived fungal genera, isolated from marine macro-organisms such as marine algae, sponges, coral, and mangrove plants, as well as deep sea sediments, species of Aspergillus were found to produce the highest number of peptides (174 peptides), followed by Penicillium (23 peptides), Acremonium (22 peptides), Eurotium (18 peptides), Trichoderma (18 peptides), Simplicillium (17 peptides), and Beauveria (12 peptides). The cytotoxic activity against a broad spectrum of human cancer cell lines was the predominant biological activity of the reported marine peptides (32%), whereas antibacterial, antifungal, antiviral, anti-inflammatory, and various enzyme inhibition activities ranged from 7% to 20%. In the first part of this review, the chemistry of marine peptides is discussed and followed by their biological activity.

Keywords:  antibacterial activity; cyclic peptides; cytotoxicity; depsipeptides; linear peptides; marine-derived fungi; marine-derived peptides

DOI:  https://doi.org/10.3390/md21100510
Membranes (Basel). 2023 Sep 28. pii: 816. [Epub ahead of print]13(10):

The Membrane-Mediated Interaction of Liquid-Ordered Lipid Domains in the Presence of Amphipathic Peptides.

Konstantin V Pinigin, Sergey A Akimov.

  The lipid membranes of living cells are composed of a large number of lipid types and can undergo phase separation with the formation of nanometer-scale liquid-ordered lipid domains, also called rafts. Raft coalescence, i.e., the fusion of lipid domains, is involved in important cell processes, such as signaling and trafficking. In this work, within the framework of the theory of elasticity of lipid membranes, we explore how amphipathic peptides adsorbed on lipid membranes may affect the domain-domain fusion processes. We show that the elastic deformations of lipid membranes drive amphipathic peptides to the boundary of lipid domains, which leads to an increase in the average energy barrier of the domain-domain fusion, even if the surface concentration of amphipathic peptides is low and the domain boundaries are only partially occupied by the peptides. This inhibition of the fusion of lipid domains may lead to negative side effects of using amphipathic peptides as antimicrobial agents.

Keywords:  amphipathic peptide; domain interaction; lipid membrane; liquid-ordered lipid domain; membrane-mediated interaction; raft coalescence; theory of elasticity

DOI:  https://doi.org/10.3390/membranes13100816
Proc Natl Acad Sci U S A. 2023 Oct 31. 120(44): e2301064120

An amino-domino model described by a cross-peptide-bond Ramachandran plot defines amino acid pairs as local structural units.

Aviv A Rosenberg, Nitsan Yehishalom, Ailie Marx, Alex M Bronstein.

  Protein structure, both at the global and local level, dictates function. Proteins fold from chains of amino acids, forming secondary structures, α-helices and β-strands, that, at least for globular proteins, subsequently fold into a three-dimensional structure. Here, we show that a Ramachandran-type plot focusing on the two dihedral angles separated by the peptide bond, and entirely contained within an amino acid pair, defines a local structural unit. We further demonstrate the usefulness of this cross-peptide-bond Ramachandran plot by showing that it captures β-turn conformations in coil regions, that traditional Ramachandran plot outliers fall into occupied regions of our plot, and that thermophilic proteins prefer specific amino acid pair conformations. Further, we demonstrate experimentally that the effect of a point mutation on backbone conformation and protein stability depends on the amino acid pair context, i.e., the identity of the adjacent amino acid, in a manner predictable by our method.

Keywords:  Ramachandran outlier; Ramachandran plot; dihedral angle; protein structure; secondary structure

DOI:  https://doi.org/10.1073/pnas.2301064120
Org Lett. 2023 Oct 27.

Synthesis of DNA-Encoded Macrocyclic Peptides via Nitrile-Aminothiol Click Reaction.

Lijun Fan, Yang Yu, Charles Jayne, John R Frost, Jack D Scott.

DNA-encoded library (DEL) technology holds exciting potential for discovering novel therapeutic macrocyclic peptides (MPs). Herein, we describe the development of a DEL-compatible peptide macrocyclization method that proceeds via intramolecular click-condensation between 3-(2-cyano-4-pyridyl)-l-alanine (Cpa) and an N-terminal cysteine. Cyclization takes place spontaneously in a buffered aqueous solution and affords the cyclized products in excellent yields. The reaction exhibits a broad substrate scope and can be employed to generate MPs of variable ring size and amino acid composition.

DOI: https://doi.org/10.1021/acs.orglett.3c03284
Commun Chem. 2023 Oct 25. 6(1): 229

Peptide binder design with inverse folding and protein structure prediction.

Patrick Bryant, Arne Elofsson.

The computational design of peptide binders towards a specific protein interface can aid diagnostic and therapeutic efforts. Here, we design peptide binders by combining the known structural space searched with Foldseek, the protein design method ESM-IF1, and AlphaFold2 (AF) in a joint framework. Foldseek generates backbone seeds for a modified version of ESM-IF1 adapted to protein complexes. The resulting sequences are evaluated with AF using an MSA representation for the receptor structure and a single sequence for the binder. We show that AF can accurately evaluate protein binders and that our bind score can select these (ROC AUC = 0.96 for the heterodimeric case). We find that designs created from seeds with more contacts per residue are more successful and tend to be short. There is a relationship between the sequence recovery in interface positions and the plDDT of the designs, where designs with ≥80% recovery have an average plDDT of 84 compared to 55 at 0%. Designed sequences have 60% higher median plDDT values towards intended receptors than non-intended ones. Successful binders (predicted interface RMSD ≤ 2 Å) are designed towards 185 (6.5%) heteromeric and 42 (3.6%) homomeric protein interfaces with ESM-IF1 compared with 18 (1.5%) using ProteinMPNN from 100 samples.

DOI: https://doi.org/10.1038/s42004-023-01029-7
Toxins (Basel). 2023 Oct 05. pii: 600. [Epub ahead of print]15(10):

Discovery of an Insect Neuroactive Helix Ring Peptide from Ant Venom.

Valentine Barassé, Laurence Jouvensal, Guillaume Boy, Arnaud Billet, Steven Ascoët, Benjamin Lefranc, Jérôme Leprince, Alain Dejean, Virginie Lacotte, Isabelle Rahioui, Catherine Sivignon, Karen Gaget, Mélanie Ribeiro Lopes, Federica Calevro, Pedro Da Silva, Karine Loth, Françoise Paquet, Michel Treilhou, Elsa Bonnafé, Axel Touchard.

  Ants are among the most abundant terrestrial invertebrate predators on Earth. To overwhelm their prey, they employ several remarkable behavioral, physiological, and biochemical innovations, including an effective paralytic venom. Ant venoms are thus cocktails of toxins finely tuned to disrupt the physiological systems of insect prey. They have received little attention yet hold great promise for the discovery of novel insecticidal molecules. To identify insect-neurotoxins from ant venoms, we screened the paralytic activity on blowflies of nine synthetic peptides previously characterized in the venom of Tetramorium bicarinatum. We selected peptide U11, a 34-amino acid peptide, for further insecticidal, structural, and pharmacological experiments. Insecticidal assays revealed that U11 is one of the most paralytic peptides ever reported from ant venoms against blowflies and is also capable of paralyzing honeybees. An NMR spectroscopy of U11 uncovered a unique scaffold, featuring a compact triangular ring helix structure stabilized by a single disulfide bond. Pharmacological assays using Drosophila S2 cells demonstrated that U11 is not cytotoxic, but suggest that it may modulate potassium conductance, which structural data seem to corroborate and will be confirmed in a future extended pharmacological investigation. The results described in this paper demonstrate that ant venom is a promising reservoir for the discovery of neuroactive insecticidal peptides.

Keywords:  Tetramorium bicarinatum; ant venoms; helix peptide; neurotoxins; potassium channel

DOI:  https://doi.org/10.3390/toxins15100600
Angew Chem Int Ed Engl. 2023 Oct 24. e202314597

Substrates and Cyclic Peptide Inhibitors of the Oligonucleotide Activated Sirtuin 7.

Julie E Bolding, Alexander L Nielsen, Iben Jensen, Tobias N Hansen, Line A Ryberg, Samuel T Jameson, Pernille Harris, Günther H J Peters, John M Denu, Joseph M Rogers, Christian Adam Olsen.

  The sirtuins are NAD+-dependent lysine deacylases, comprising seven isoforms (SIRT1-7) in humans, which are involved in the regulation of a plethora of biological processes, including gene expression and metabolism. The sirtuins share a common hydrolytic mechanism but display preferences for different ε-N-acyllysine substrates. SIRT7 deacetylates targets in nuclei and nucleoli but remains one of the lesser studied of the seven isoforms; in part, due to a lack of chemical tools to specifically probe SIRT7 activity. Here we expressed SIRT7 and, using small-angle X-ray scattering, reveal SIRT7 to be a monomeric enzyme with low degree of globular flexibility in solution. We developed a fluorogenic assay for investigation of the substrate preferences of SIRT7 and to evaluate compounds that modulate its activity. We report several mechanism-based SIRT7 inhibitors as well as de novo cyclic peptide inhibitors selected from mRNA-display library screening that exhibit selectivity for SIRT7 over other sirtuin isoforms, stabilize SIRT7 in cells, and cause increase in the acetylation H3K18.

Keywords:  Sirtuins; epigenetics; histones; mRNA display; posttranslational modification

DOI:  https://doi.org/10.1002/anie.202314597
Molecules. 2023 Oct 16. pii: 7118. [Epub ahead of print]28(20):

Tandem Mass Spectrometry de novo Sequencing of the Skin Defense Peptides of the Central Slovenian Agile Frog Rana dalmatina.

Tatiana Yu Samgina, Irina D Vasileva, Polonca Trebše, Gregor Torkar, Alexey K Surin, Zhaowei Meng, Roman A Zubarev, Albert T Lebedev.

  Peptides released on frogs' skin in a stress situation represent their only weapon against micro-organisms and predators. Every species and even population of frog possesses its own peptidome being appropriate for their habitat. Skin peptides are considered potential pharmaceuticals, while the whole peptidome may be treated as a taxonomic characteristic of each particular population. Continuing the studies on frog peptides, here we report the peptidome composition of the Central Slovenian agile frog Rana dalmatina population. The detection and top-down de novo sequencing of the corresponding peptides was conducted exclusively by tandem mass spectrometry without using any chemical derivatization procedures. Collision-induced dissociation (CID), higher energy collision-induced dissociation (HCD), electron transfer dissociation (ETD) and combined MS3 method EThcD with stepwise increase of HCD energy were used for that purpose. MS/MS revealed the whole sequence of the detected peptides including differentiation between isomeric Leu/Ile, and the sequence portion hidden in the disulfide cycle. The array of the discovered peptide families (brevinins 1 and 2, melittin-related peptides (MRPs), temporins and bradykinin-related peptides (BRPs)) is quite similar to that of R. temporaria. Since the genome of this frog remains unknown, the obtained results were compared with the recently published transcriptome of R. dalmatina.

Keywords:  EThcD; agile brown frog; bradykinin-related peptides (BRPs); membrane-active peptides; tandem mass spectrometry; top-down de novo sequencing

DOI:  https://doi.org/10.3390/molecules28207118
Cancers (Basel). 2023 Oct 20. pii: 5071. [Epub ahead of print]15(20):

Compromised Blood-Brain Barrier Junctions Enhance Melanoma Cell Intercalation and Extravasation.

Federico Saltarin, Adrian Wegmüller, Leire Bejarano, Ece Su Ildiz, Pascale Zwicky, Andréj Vianin, Florentin Spadin, Klara Soukup, Vladimir Wischnewski, Britta Engelhardt, Urban Deutsch, Ines J Marques, Martin Frenz, Johanna A Joyce, Ruth Lyck.

  Melanoma frequently metastasises to the brain, and a detailed understanding of the molecular and cellular mechanisms underlying melanoma cell extravasation across the blood-brain barrier (BBB) is important for preventing brain metastasis formation. Making use of primary mouse brain microvascular endothelial cells (pMBMECs) as an in vitro BBB model, we imaged the interaction of melanoma cells into pMBMEC monolayers. We observed exclusive junctional intercalation of melanoma cells and confirmed that melanoma-induced pMBMEC barrier disruption can be rescued by protease inhibition. Interleukin (IL)-1β stimulated pMBMECs or PECAM-1-knockout (-ko) pMBMECs were employed to model compromised BBB barrier properties in vitro and to determine increased melanoma cell intercalation compared to pMBMECs with intact junctions. The newly generated brain-homing melanoma cell line YUMM1.1-BrM4 was used to reveal increased in vivo extravasation of melanoma cells across the BBB of barrier-compromised PECAM-1-deficient mice compared to controls. Taken together, our data indicate that preserving BBB integrity is an important measure to limit the formation of melanoma-brain metastasis.

Keywords:  blood-brain barrier (BBB); cell extravasation; endothelial junctions; melanoma; metastasis

DOI:  https://doi.org/10.3390/cancers15205071
Cells. 2023 Oct 18. pii: 2474. [Epub ahead of print]12(20):

Long Prehensile Protrusions Can Facilitate Cancer Cell Invasion through the Basement Membrane.

Shayan S Nazari, Andrew D Doyle, Christopher K E Bleck, Kenneth M Yamada.

  A basic process in cancer is the breaching of basement-membrane barriers to permit tissue invasion. Cancer cells can use proteases and physical mechanisms to produce initial holes in basement membranes, but how cells squeeze through this barrier into matrix environments is not well understood. We used a 3D invasion model consisting of cancer-cell spheroids encapsulated by a basement membrane and embedded in collagen to characterize the dynamic early steps in cancer-cell invasion across this barrier. We demonstrate that certain cancer cells extend exceptionally long (~30-100 μm) protrusions through basement membranes via actin and microtubule cytoskeletal function. These long protrusions use integrin adhesion and myosin II-based contractility to pull cells through the basement membrane for initial invasion. Concurrently, these long, organelle-rich protrusions pull surrounding collagen inward while propelling cancer cells outward through perforations in the basement-membrane barrier. These exceptionally long, contractile cellular protrusions can facilitate the breaching of the basement-membrane barrier as a first step in cancer metastasis.

Keywords:  3D culture; basement membrane; cell protrusion; collagen; contractility; cytoskeleton; integrin; invasion; myosin II; spheroid

DOI:  https://doi.org/10.3390/cells12202474