bims-cepepe 2024-08-04 papers

bims-cepepe

Biomed News

on Cell-penetrating peptides

Issue of 2024–08–04
seventeen papers selected by
Henry Lamb, Queensland University of Technology

A facile strategy for the construction of a phage display cyclic peptide library for the selection of functional macrocycles.
MuCoCP: A priori chemical knowledge-based multimodal contrastive learning pre-trained neural network for the prediction of cyclic peptide membrane penetration ability.
Hijacking of N-fixing legume albumin-1 genes enables the cyclization and stabilization of defense peptides.
De Novo Design of Peptide Binders to Conformationally Diverse Targets with Contrastive Language Modeling.
Deep mutational scanning and machine learning for the analysis of antimicrobial-peptide features driving membrane selectivity.
Relationship between oligoarginine-induced membrane damage of single Escherichia coli cells and entry of the peptide into the cytoplasm.
PICKAPEP: An application for parameter calculation and visualization of cyclized and modified peptidomimetics.
Discovery of novel penetrating peptides able to target human leukemia and lymphoma for enhanced PROTAC delivery.
Enhancing the Intrinsic Antiplasmodial Activity and Improving the Stability and Selectivity of a Tunable Peptide Scaffold Derived from Human Platelet Factor 4.
Intracellular delivery strategies using membrane-interacting peptides and proteins.
Selection and characterization of a peptide-based complement modulator targeting C1 of the innate immune system.
Effects of PEGylation on Imaging Contrast of 68Ga-Labeled Bicyclic Peptide PET Probes Targeting Nectin-4.
Identification of the Clinical Candidate PF-07284890 (ARRY-461), a Highly Potent and Brain Penetrant BRAF Inhibitor for the Treatment of Cancer.
Organelle-Targeted Laurdans Measure Heterogeneity in Subcellular Membranes and Their Responses to Saturated Lipid Stress.
Protocol for delivering proteins, peptides, and shRNAs via the intrathecal route in the experimental allergic encephalomyelitis mouse model.
Synergistic anticancer immunity in metastatic triple-negative breast cancer through an in situ amplifying Peptide-Drug Conjugate.
Diffusing protein binders to intrinsically disordered proteins.

Chem Sci. 2024 Jul 31. 15(30): 11847-11855

A facile strategy for the construction of a phage display cyclic peptide library for the selection of functional macrocycles.

Hua Xiang, Liwen Bai, Xindan Zhang, Ting Dan, Peng Cheng, Xiaoqin Yang, Honglian Ai, Kai Li, Xinxiang Lei.

Cyclic peptides represent invaluable scaffolds in biological affinity, providing diverse collections for discovering functional molecules targeting challenging biological entities and protein-protein interactions. The field increasingly focuses on developing cyclization strategies and chemically modified combinatorial libraries in conjunction with M13 phage display, to identify macrocyclic peptide inhibitors for traditionally challenging targets. Here, we introduce a cyclization strategy utilizing ortho-phthalaldehyde (OPA) for the discovery of active macrocycles characterized by asymmetric scaffolds with side-chain cyclization. Through this approach, aldehyde groups attached to free molecules sequentially attack the ε-amine of lysine and the thiol of cysteine, facilitating the rapid cyclization of genetically encoded linear precursor libraries displayed on phage particles. The construction of a 109-member library and subsequent screening successfully identified cyclic peptide binders targeting three therapeutically relevant proteins: PTP1B, NEK7, and hKeap1. The results confirm the efficacy in rapidly obtaining active ligands with micromolar potency. This work provides a fast and efficient operable high-throughput platform for screening functional peptide macrocycles, which hold promise for broad application in therapeutics, chemically biological probes, and disease diagnosis.

DOI: https://doi.org/10.1039/d4sc03207a
Bioinformatics. 2024 Jul 27. pii: btae473. [Epub ahead of print]

MuCoCP: A priori chemical knowledge-based multimodal contrastive learning pre-trained neural network for the prediction of cyclic peptide membrane penetration ability.

Yunxiang Yu, Mengyun Gu, Hai Guo, Yabo Deng, Danna Chen, Jianwei Wang, Caixia Wang, Xia Liu, Wenjin Yan, Jinqi Huang.

   MOTIVATION: There has been a burgeoning interest in cyclic peptide therapeutics due to their various outstanding advantages and strong potential for drug formation. However, it is undoubtedly costly and inefficient to use traditional wet lab methods to clarify their biological activities. Using Artificial Intelligence instead is a more energy-efficient and faster approach. MuCoCP aims to build a complete pre-trained model for extracting potential features of cyclic peptides, which can be fine-tuned to accurately predict cyclic peptide bioactivity on various downstream tasks. To maximize its effectiveness, we use a novel data augmentation method based on a priori chemical knowledge and multiple unsupervised training objective functions to greatly improve the information-grabbing ability of the model.
RESULTS: To assay the efficacy of the model, we conducted validation on the membrane-permeability of cyclic peptides which achieved an accuracy of 0.87 and R-squared of 0.503 on CycPeptMPDB using semi-supervised training and obtained an accuracy of 0.84 and R-squared of 0.384 using a model with frozen parameters on an external dataset. This result has achieved state-of-the-art (SOTA), which substantiates the stability and generalization capability of MuCoCP. It means that MuCoCP can fully explore the high-dimensional information of cyclic peptides and make accurate predictions on downstream bioactivity tasks, which will serve as a guide for the future de novo design of cyclic peptide drugs and promote the development of cyclic peptide drugs.
AVAILABILITY: All code used in our proposed method can be found at https://github.com/lennonyu11234/MuCoCP.
SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics.

Keywords:  Contrastive learning; Cyclic Peptides; Deep multimodal neural network; permeability

DOI:  https://doi.org/10.1093/bioinformatics/btae473
Nat Commun. 2024 Aug 03. 15(1): 6565

Hijacking of N-fixing legume albumin-1 genes enables the cyclization and stabilization of defense peptides.

Edward K Gilding, Mark A Jackson, Linh T T Nguyen, Brett R Hamilton, Katherine A Farquharson, Wing L Ho, Kuok Yap, Carolyn J Hogg, Katherine Belov, David J Craik.

The legume albumin-1 gene family, arising after nodulation, encodes linear a- and b-chain peptides for nutrient storage and defense. Intriguingly, in one prominent legume, Clitoria ternatea, the b-chains are replaced by domains producing ultra-stable cyclic peptides called cyclotides. The mechanism of this gene hijacking is until now unknown. Cyclotides require recruitment of ligase-type asparaginyl endopeptidases (AEPs) for maturation (cyclization), necessitating co-evolution of two gene families. Here we compare a chromosome-level C. ternatea genome with grain legumes to reveal an 8 to 40-fold expansion of the albumin-1 gene family, enabling the additional loci to undergo diversification. Iterative rounds of albumin-1 duplication and diversification create four albumin-1 enriched genomic islands encoding cyclotides, where they are physically grouped by similar pI and net charge values. We identify an ancestral hydrolytic AEP that exhibits neofunctionalization and multiple duplication events to yield two ligase-type AEPs. We propose cyclotides arise by convergence in C. ternatea where their presence enhances defense from biotic attack, thus increasing fitness compared to lineages with linear b-chains and ultimately driving the replacement of b-chains with cyclotides.

DOI: https://doi.org/10.1038/s41467-024-50742-x
bioRxiv. 2024 Jul 22. pii: 2023.06.26.546591. [Epub ahead of print]

De Novo Design of Peptide Binders to Conformationally Diverse Targets with Contrastive Language Modeling.

Suhaas Bhat, Kalyan Palepu, Lauren Hong, Joey Mao, Tianzheng Ye, Rema Iyer, Lin Zhao, Tianlai Chen, Sophia Vincoff, Rio Watson, Tian Wang, Divya Srijay, Venkata Srikar Kavirayuni, Kseniia Kholina, Shrey Goel, Pranay Vure, Aniruddha J Desphande, Scott H Soderling, Matthew P DeLisa, Pranam Chatterjee.

Designing binders to target undruggable proteins presents a formidable challenge in drug discovery, requiring innovative approaches to overcome the lack of putative binding sites. Recently, generative models have been trained to design binding proteins via three-dimensional structures of target proteins, but as a result, struggle to design binders to disordered or conformationally unstable targets. In this work, we provide a generalizable algorithmic framework to design short, target-binding linear peptides, requiring only the amino acid sequence of the target protein. To do this, we propose a process to generate naturalistic peptide candidates through Gaussian perturbation of the peptidic latent space of the ESM-2 protein language model, and subsequently screen these novel linear sequences for target-selective interaction activity via a CLIP-based contrastive learning architecture. By integrating these generative and discriminative steps, we create a Pep tide Pr ioritization via CLIP ( PepPrCLIP ) pipeline and validate highly-ranked, target-specific peptides experimentally, both as inhibitory peptides and as fusions to E3 ubiquitin ligase domains, demonstrating functionally potent binding and degradation of conformationally diverse protein targets in vitro . Overall, our design strategy provides a modular toolkit for designing short binding linear peptides to any target protein without the reliance on stable and ordered tertiary structure, enabling generation of programmable modulators to undruggable and disordered proteins such as transcription factors and fusion oncoproteins.

DOI: https://doi.org/10.1101/2023.06.26.546591
Nat Biomed Eng. 2024 Jul 31.

Deep mutational scanning and machine learning for the analysis of antimicrobial-peptide features driving membrane selectivity.

Justin R Randall, Luiz C Vieira, Claus O Wilke, Bryan W Davies.

Many antimicrobial peptides directly disrupt bacterial membranes yet can also damage mammalian membranes. It is therefore central to their therapeutic use that rules governing the membrane selectivity of antimicrobial peptides be deciphered. However, this is difficult even for short peptides owing to the large combinatorial space of amino acid sequences. Here we describe a method for measuring the loss or maintenance of antimicrobial-peptide activity for thousands of peptide-sequence variants simultaneously, and its application to Protegrin-1, a potent yet toxic antimicrobial peptide, to determine the positional importance and flexibility of residues across its sequence while identifying variants with changes in membrane selectivity. More bacterially selective variants maintained a membrane-bound secondary structure while avoiding aromatic residues and cysteine pairs. A machine-learning model trained with our datasets accurately predicted membrane-specific activities for over 5.7 million Protegrin-1 variants, and identified one variant that showed substantially reduced toxicity and retention of activity in a mouse model of intraperitoneal infection. The high-throughput methodology may help elucidate sequence-structure-function relationships in antimicrobial peptides and inform the design of peptide-based synthetic drugs.

DOI: https://doi.org/10.1038/s41551-024-01243-1
Biochem Biophys Rep. 2024 Sep;39 101777

Relationship between oligoarginine-induced membrane damage of single Escherichia coli cells and entry of the peptide into the cytoplasm.

Sabrina Sharmin, Md Zahidul Islam, Masahito Yamazaki.

  Cell-penetrating peptides (CPPs) can enter the cytosol of eukaryotic cells without killing them whereas some CPPs exhibit antimicrobial activity against bacterial cells. Here, to elucidate the mode of interaction of the CPP nona-arginine (R9) with bacterial cells, we investigated the interactions of lissamine rhodamine B red-labeled peptide (Rh-R9) with single Escherichia coli cells encapsulating calcein using confocal laser scanning microscopy. After Rh-R9 induced the leakage of a large amount of calcein, the fluorescence intensity of the cytosol due to Rh-R9 greatly increased, indicating that Rh-R9 induces cell membrane damage, thus allowing entry of a significant amount of Rh-R9 into the cytosol. To determine if the lipid bilayer region of the membrane is the main target of Rh-R9, we then investigated the interaction of Rh-R9 with single giant unilamellar vesicles (GUVs) comprising an E. coli polar lipid extract containing small GUVs and AlexaFluor 647 hydrazide (AF647) in the lumen. Rh-R9 entered the GUV lumen without inducing AF647 leakage, but leakage eventually did occur, indicating that GUV membrane damage was induced after the entry of Rh-R9 into the GUV lumen. The Rh-R9 peptide concentration dependence of the fraction of entry of Rh-R9 after a specific interaction time was similar to that of the fraction of leaking GUVs. These results indicate that Rh-R9 can damage the lipid bilayer region of a cell membrane, which may be related to its antimicrobial activity.

Keywords:  Antimicrobial activity; Cell-penetrating peptides; E. coli cells; Entry of peptides; Giant unilamellar vesicles; Membrane damage

DOI:  https://doi.org/10.1016/j.bbrep.2024.101777
J Pept Sci. 2024 Jul 31. e3646

PICKAPEP: An application for parameter calculation and visualization of cyclized and modified peptidomimetics.

Vanessa Erckes, Mattis Hilleke, Clemens Isert, Christian Steuer.

  The interest in peptides and especially in peptidomimetic structures has risen enormously in the past few years. Novel modification strategies including nonnatural amino acids, sophisticated cyclization strategies, and side chain modifications to improve the pharmacokinetic properties of peptides are continuously arising. However, a calculator tool accompanying the current development in peptide sciences towards modified peptides is missing. Herein, we present the application PICKAPEP, enabling the virtual construction and visualization of peptidomimetics ranging from well-known cyclized and modified peptides such as ciclosporin A up to fully self-designed peptide-based structures with custom amino acids. Calculated parameters include the molecular weight, the water-octanol partition coefficient, the topological polar surface area, the number of rotatable bonds, and the peptide SMILES code. To our knowledge, PICKAPEP is the first tool allowing users to add custom amino acids as building blocks and also the only tool giving the possibility to process large peptide libraries and calculate parameters for multiple peptides at once. We believe that PICKAPEP will support peptide researchers in their work and will find wide application in current as well as future peptide drug development processes. PICKAPEP is available open source for Windows and Mac operating systems (https://urldefense.com/v3/__https://www.research-collection.ethz.ch/handle/20.500.11850/681174__;!!N11eV2iwtfs!qt5f_2lNd6IZUDH1HVSVwg0zYzS8-nFazQ8c61jS5GaD5vkVS5C3igyfh3haJRnaX8ugW7o9VWUiCihPqcptmaWoqwYf9LvZTQ$).

Keywords:  cyclization; library processing; modification; peptides; peptidomimetics; physicochemical properties; software tool

DOI:  https://doi.org/10.1002/psc.3646
Eur J Med Chem. 2024 Jul 31. pii: S0223-5234(24)00615-9. [Epub ahead of print]277 116734

Discovery of novel penetrating peptides able to target human leukemia and lymphoma for enhanced PROTAC delivery.

Qingqing Zhang, Yuying Liu, Jie Zhang, Yanchen Li, Jin Wang, Nanxin Liu, Jie Zhang, Xiaoyan Pan.

  Proteolysis targeting chimeras (PROTAC) are bifunctional chimeric molecules capable of directly degrading binding proteins through the ubiquitin-proteasome pathway. PROTACs have demonstrated significant potential in overcoming drug resistance and targeting previously untreatable targets. However, several limitations still need to be addressed, including their high molecular weight resulting in poor membrane permeability and bioavailability. In this study, we proposed that cancer-targeted penetrating peptides could enhance the cell permeability of PROTACs. We developed 26 novel targeted penetrating peptides for leukemia and lymphoma cells, among which C9C-f(3Bta) and Cyclo-C9C-R exhibited superior membrane permeability, targetability, and stability. By combining C9C-f(3Bta) and Cyclo-C9C-R with IMA-PROTAC, we effectively enhanced the anti-proliferative activity of IMA-PROTAC, facilitated degradation of Bcr-Abl protein in K562 cells, and reduced downstream STAT5 phosphorylation. Furthermore, the combined application promoted cell apoptosis while blocking G1 phase progression. HPLC-MRM-MS revealed that the combination of C9C-f(3Bta) or Cyclo-C9C-R with IMA-PROTAC significantly enhanced intracellular IMA-PROTAC content. In summary, our proof-of-concept study validated the hypothesis that combining PROTACs with targeted penetrating peptides can improve protein degradation efficiency as well as anti-proliferative capabilities.

Keywords:  Antitumor; Cancer-targeted penetrating peptide (CTP); Leukemia treatment; PROTAC; Targeted delivery

DOI:  https://doi.org/10.1016/j.ejmech.2024.116734
ACS Infect Dis. 2024 Aug 01.

Enhancing the Intrinsic Antiplasmodial Activity and Improving the Stability and Selectivity of a Tunable Peptide Scaffold Derived from Human Platelet Factor 4.

Nicole Lawrence, Thomas N G Handley, Simon J de Veer, Maxim D Harding, Alicja Andraszek, Lachlan Hall, Karoline D Raven, Sandra Duffy, Vicky M Avery, David J Craik, Lara R Malins, Brendan J McMorran.

  The control of malaria, a disease caused by Plasmodium parasites that kills over half a million people every year, is threatened by the continual emergence and spread of drug resistance. Therefore, new molecules with different mechanisms of action are needed in the antimalarial drug development pipeline. Peptides developed from host defense molecules are gaining traction as anti-infectives due to theood of inducing drug resistance. Human platelet factor 4 (PF4) has intrinsic activity against P. falciparum, and a macrocyclic helix-loop-helix peptide derived from its active domain recapitulates this activity. In this study, we used a stepwise approach to optimize first-generation PF4-derived internalization peptides (PDIPs) by producing analogues with substitutions to charged and hydrophobic amino acid residues or with modifications to terminal residues including backbone cyclization. We evaluated the in vitro activity of PDIP analogues against P. falciparum compared to their overall helical structure, resistance to breakdown by serum proteases, selective binding to negatively charged membranes, and hemolytic activity. Next, we combined antiplasmodial potency-enhancing substitutions that retained favorable membrane and cell-selective properties onto the most stable scaffold to produce a backbone cyclic PDIP analogue with four-fold improved activity against P. falciparum compared to first-generation peptides. These studies demonstrate the ability to modify PDIP to select for and combine desirable properties and further validate the suitability of this unique peptide scaffold for developing a new molecule class that is distinct from existing antimalarial drugs.

Keywords:  Plasmodium; drug development; host defense peptide; malaria; rational design; targeted cell-penetration

DOI:  https://doi.org/10.1021/acsinfecdis.4c00276
Nanoscale. 2024 Aug 02.

Intracellular delivery strategies using membrane-interacting peptides and proteins.

Linh D Mai, Sydney C Wimberley, Julie A Champion.

While the cellular cytosol and organelles contain attractive targets for disease treatments, it remains a challenge to deliver therapeutic biomacromolecules to these sites. This is due to the selective permeability of the plasma and endosomal membranes, especially for large and hydrophilic therapeutic cargos such as proteins and nucleic acids. In response, many different delivery systems and molecules have been devised to help therapeutics cross these barriers to reach cytosolic targets. Among them are peptide and protein-based systems, which have several advantages over other natural and synthetic materials including their ability to interact with cell membranes. In this review, we will describe recent advances and current challenges of peptide and protein strategies that leverage cell membrane association and modulation to enable cytosolic delivery of biomacromolecule cargo. The approaches covered here include peptides and proteins derived from or inspired by natural sequences as well as those designed de novo for delivery function.

DOI: https://doi.org/10.1039/d4nr02093f
RSC Chem Biol. 2024 Jul 31. 5(8): 787-799

Selection and characterization of a peptide-based complement modulator targeting C1 of the innate immune system.

Sebastiaan M W R Hamers, Leoni Abendstein, Aimee L Boyle, Seino A K Jongkees, Thomas H Sharp.

The human complement pathway plays a pivotal role in immune defence, homeostasis, and autoimmunity regulation, and complement-based therapeutics have emerged as promising interventions, with both antagonistic and agonistic approaches being explored. The classical pathway of complement is initiated when the C1 complex binds to hexameric antibody platforms. Recent structural data revealed that C1 binds to small, homogeneous interfaces at the periphery of the antibody platforms. Here, we have developed a novel strategy for complement activation using macrocyclic peptides designed to mimic the interface between antibodies and the C1 complex. In vitro selection utilizing the RaPID system identified a cyclic peptide (cL3) that binds to the C1 complex via the globular head domains of C1q. Notably, when immobilized on surfaces, cL3 effectively recruits C1 from human serum, activates C1s proteases, and induces lysis of cell-mimetic lipid membranes. This represents the first instance of a peptide capable of activating complement by binding C1 when immobilized. Further characterization and synthesis of deletion mutants revealed a critical cycle size of cL3 essential for C1 binding and efficient complement activation. Importantly, cL3 also demonstrated the ability to inhibit complement-mediated lysis without affecting C1 binding, highlighting its potential as a therapeutic modality to prevent complement-dependent cytotoxicity whilst promoting cellular phagocytosis and cell clearance. In summary, this study introduces the concept of "Peptactins" - peptide-based activators of complement - and underscores the potential of macrocyclic peptides for complement modulation, offering potential advantages over traditional biologicals in terms of size, production, and administration.

DOI: https://doi.org/10.1039/d4cb00081a
Mol Pharm. 2024 Jul 29.

Effects of PEGylation on Imaging Contrast of 68Ga-Labeled Bicyclic Peptide PET Probes Targeting Nectin-4.

Qiang Wan, Hongmei Yuan, Ping Cai, Yang Liu, Ting Yan, Li Wang, Zhijun Zhou, Wei Zhang, Nan Liu.

  Nectin cell adhesion molecule 4 (Nectin-4) is overexpressed in various malignant tumors and has emerged as a promising target for tumor imaging. Bicyclic peptides, known for their conformational rigidity, metabolic stability, and membrane permeability, are ideal tracers for positron emission tomography (PET) imaging. In this study, we evaluated the feasibility of visualizing Nectin-4-positive tumors using radiolabeled bicyclic peptide derivatives and optimized the pharmacokinetics of radiotracers by introducing PEG chains of different lengths. Five PEGylated radiotracers radiolabeled with 68Ga3+ exhibited high radiochemical purity and stability. As the chain length increased, the Log D values decreased from -2.32 ± 0.13 to -2.50 ± 0.16, indicating a gradual increase in the hydrophilicity of the radiotracers. In vitro cell-binding assay results showed that the PEGylated bicyclic peptide exhibits nanomolar affinity, and blocking experiments confirmed the specific binding of the tracers to the Nectin-4 receptor. In vivo PET imaging and biodistribution studies in SW780 and 5637 xenograft mice showed that [68Ga]Ga-NOTA-PEG12-BP demonstrated optimal pharmacokinetics, characterized by rapid and good tumor uptake, faster background clearance, and improved tumor-to-tissue contrast. Finally, compared with 18F-FDG, PET imaging, in vivo blocking assays of [68Ga]Ga-NOTA-PEG12-BP and histological staining confirmed that specific tumor uptake was mediated by Nectin-4 receptors. The results indicated that [68Ga]Ga-NOTA-PEG12-BP was a promising PET radiotracer for Nectin-4 targeting, with applications for clinical translation.

Keywords:  Nectin-4; PEGylation; PET imaging; bicyclic peptide; targeting tracer

DOI:  https://doi.org/10.1021/acs.molpharmaceut.4c00366
J Med Chem. 2024 Jul 30.

Identification of the Clinical Candidate PF-07284890 (ARRY-461), a Highly Potent and Brain Penetrant BRAF Inhibitor for the Treatment of Cancer.

Li Ren, David Moreno, Brian R Baer, Patrick Barbour, Tanna Bettendorf, Karyn Bouhana, Karin Brown, Suzy A Brown, Jay B Fell, Dylan P Hartley, Erik J Hicken, Ellen R Laird, Patrice Lee, Joseph McCown, Jennifer N Otten, Brett Prigaro, Ross Wallace, Dean Kahn.

Mutant BRAFV600E is one of the most common oncogenic drivers in metastatic melanoma. While first generation BRAFV600E inhibitors are capable of controlling tumors systemically, they are unable to adequately treat tumors that have metastasized to the brain due to insufficient penetration across the blood-brain barrier (BBB). Through a combination of structure-based drug design (SBDD) and the optimization of physiochemical properties to enhance BBB penetration, we herein report the discovery of the brain-penetrant BRAFV600E inhibitor PF-07284890 (ARRY-461). In mice studies, ARRY-461 proved to be highly brain-penetrant and was able to drive regressions of A375 BRAFV600E tumors implanted both subcutaneously and intracranially. Based on compelling preclinical safety and efficacy studies, ARRY-461 was progressed into a Phase 1 A/B clinical trial (NCT04543188).

DOI: https://doi.org/10.1021/acs.jmedchem.4c00998
ACS Chem Biol. 2024 Jul 28.

Organelle-Targeted Laurdans Measure Heterogeneity in Subcellular Membranes and Their Responses to Saturated Lipid Stress.

Adrian M Wong, Itay Budin.

Organelles feature characteristic lipid compositions that lead to differences in membrane properties. In cells, membrane ordering and fluidity are commonly measured using the solvatochromic dye Laurdan, whose fluorescence is sensitive to lipid packing. As a general lipophilic dye, Laurdan stains all hydrophobic environments in cells; therefore, it is challenging to characterize membrane properties in specific organelles or assess their responses to pharmacological treatments in intact cells. Here, we describe the synthesis and application of Laurdan-derived probes that read out the membrane packing of individual cellular organelles. The set of organelle-targeted Laurdans (OTL) localizes to the ER, mitochondria, lysosomes, and Golgi compartments with high specificity while retaining the spectral resolution needed to detect biological changes in membrane ordering. We show that ratiometric imaging with OTLs can resolve membrane heterogeneity within organelles as well as changes in lipid packing resulting from inhibition of trafficking or bioenergetic processes. We apply these probes to characterize organelle-specific responses to saturated lipid stress. While the ER and lysosomal membrane fluidity is sensitive to exogenous saturated fatty acids, that of mitochondrial membranes is protected. We then use differences in ER membrane fluidity to sort populations of cells based on their fatty acid diet, highlighting the ability of organelle-localized solvatochromic probes to distinguish between cells based on their metabolic state. These results expand the repertoire of targeted membrane probes and demonstrate their application in interrogating lipid dysregulation.

DOI: https://doi.org/10.1021/acschembio.4c00249
STAR Protoc. 2024 Jul 27. pii: S2666-1667(24)00384-8. [Epub ahead of print]5(3): 103219

Protocol for delivering proteins, peptides, and shRNAs via the intrathecal route in the experimental allergic encephalomyelitis mouse model.

Anup Bhusal, Kyoungho Suk.

  The blood-brain barrier hinders drug delivery to the central nervous system (CNS), particularly for large molecules. Here, we present a protocol for delivering proteins, peptides, and short hairpin RNAs (shRNAs) via the intrathecal (IT) route in the experimental allergic encephalomyelitis (EAE) mouse model. We describe steps for developing EAE in mice and administering treatments intrathecally. The insights into treatment efficacy that can be provided by this protocol make it an important tool for CNS research. For complete details on the use and execution of this protocol, please refer to Bhusal et al.1.

Keywords:  Immunology; Microscopy; Neuroscience

DOI:  https://doi.org/10.1016/j.xpro.2024.103219
J Control Release. 2024 Jul 31. pii: S0168-3659(24)00532-7. [Epub ahead of print]

Synergistic anticancer immunity in metastatic triple-negative breast cancer through an in situ amplifying Peptide-Drug Conjugate.

Ha Rin Kim, Seong Jin Park, Young Seok Cho, Yoon Gun Ko, Sang Yoon Kim, Youngro Byun.

  Despite significant progress in combining cancer immunotherapy with chemotherapy to treat triple negative breast cancer (TNBC), challenges persist due to target depletion and tumor heterogeneity, especially in metastasis. Chemotherapy lacks precise targeting abilities, and targeted therapy is inadequate in addressing the diverse heterogeneity of tumors. To address these challenges, we introduce RGDEVD-DOX as a tumor-specific immunogenic agent, namely TPD1, which targets integrin αvβ3 and gets continuously activated by apoptosis. TPD1 facilitates the caspase-3-mediated in situ amplification that results in tumor-specific accumulation of doxorubicin. This local concentration of doxorubicin induces immunogenic cell death and promotes the recruitment of immune cells to the tumor site. Notably, the tumor-targeting capabilities of TPD1 help bypass the systemic immunotoxicity of doxorubicin. Consequently, this alters the tumor microenvironment, converting it into a 'hot' tumor that is more susceptible to immune checkpoint inhibition. We demonstrated the anti-metastatic and anti-cancer efficacy of this treatment using various xenograft and metastatic models. This study underscores the high potential of caspase-3 cleavable peptide-drug conjugates to be used in conjunction with anti-cancer immunotherapies.

Keywords:  Peptide-Drug Conjugate; cancer immunotherapy; caspase-3; integrin αvβ3; metastatic triple-negative breast cancer; αPD-1 antibody

DOI:  https://doi.org/10.1016/j.jconrel.2024.07.074
bioRxiv. 2024 Jul 16. pii: 2024.07.16.603789. [Epub ahead of print]

Diffusing protein binders to intrinsically disordered proteins.

Caixuan Liu, Kejia Wu, Hojun Choi, Hannah Han, Xulie Zhang, Joseph L Watson, Sara Shijo, Asim K Bera, Alex Kang, Evans Brackenbrough, Brian Coventry, Derrick R Hick, Andrew N Hoofnagle, Ping Zhu, Xingting Li, Justin Decarreau, Stacey R Gerben, Wei Yang, Xinru Wang, Mila Lamp, Analisa Murray, Magnus Bauer, David Baker.

  Proteins which bind intrinsically disordered proteins (IDPs) and intrinsically disordered regions (IDRs) with high affinity and specificity could have considerable utility for therapeutic and diagnostic applications. However, a general methodology for targeting IDPs/IDRs has yet to be developed. Here, we show that starting only from the target sequence of the input, and freely sampling both target and binding protein conformation, RFdiffusion can generate binders to IDPs and IDRs in a wide range of conformations. We use this approach to generate binders to the IDPs Amylin, C-peptide and VP48 in a range of conformations with Kds in the 3 -100nM range. The Amylin binder inhibits amyloid fibril formation and dissociates existing fibers, and enables enrichment of amylin for mass spectrometry-based detection. For the IDRs G3bp1, common gamma chain (IL2RG) and prion, we diffused binders to beta strand conformations of the targets, obtaining 10 to 100 nM affinity. The IL2RG binder colocalizes with the receptor in cells, enabling new approaches to modulating IL2 signaling. Our approach should be widely useful for creating binders to flexible IDPs/IDRs spanning a wide range of intrinsic conformational preferences.

Keywords:  Amyloid fibril dissociation; Intrinsically disordered protein; RFdiffusion; Rosetta; deep learning; diagnostics; intrinsically disordered region; protein design

DOI:  https://doi.org/10.1101/2024.07.16.603789