Biology subjects

Teesalu, T.

Publications and source records attributed to Teesalu, T..

14 recordsLinked to original sources

bioRxiv2026

Brain-specific PP1/LRRK2 interaction-targeting peptides as a novel strategy for Parkinson's disease treatment

The LRRK2 kinase has emerged as a priority therapeutic target due to its well-documented role in Parkinson-s diseases (PD). One of LRRK2s key partners is the phosphatase PP1, which dephosphorylates LRRK2. Therefore, modulating this protein/protein interaction represents a promising therapeutic strategy for PD. We have developed a bi-functional peptide, PEP 3, capable of crossing the blood-brain barrier (BBB) and disrupting the LRRK2/PP1 interaction. In vitro competition assays confirmed that PEP 3 specifically targets this interaction. The in vivo imaging demonstrated that the peptide remains detectable in the mouse brain up to 6 hours or retro-orbital vein post-injection. The PEP 3 peptide does not show chronic toxicity in CD1 mice and is resistant to degradation by mouse, human, dog and monkey serum proteases. In mouse models overexpressing alpha-synuclein, repeated intraperitoneal injections of PEP 3 for 15 and 30 days were well tolerated. Immunohistochemistry revealed a significant reduction of dopaminergic cell death in the substantia nigra, and quantification of phosphorylated pathological alpha-synuclein showed decreased levels in the PEP 3-treated group compared to controls. These findings support the potential of PEP 3 as a therapeutic agent for Parkinsons disease.

neuroscience↗

bioRxiv2025

Proximity labelling-based identification of vascular homing peptide receptors

Identifying the receptors of vascular homing peptides (VHPs) is critical for mechanistic understanding and development of peptide-guided precision therapeutics. Conventional receptor discovery methods, such as affinity chromatography, require cell disruption and often expose intracellular proteins, resulting in high background and low specificity. To overcome these limitations, we developed a proximity labelling approach that tags proteins near VHP receptors on intact live cells. Cells were incubated with VHP-horseradish peroxidase (HRP) complexes, which, upon hydrogen peroxide treatment, activate biotin-tyramide to produce short-lived radicals that covalently label nearby membrane proteins. Using the prototypic C-end Rule peptide RPARPAR and its known receptor neuropilin-1 (NRP-1), we validated this method by successful receptor tagging and mass spectrometric identification. Using RPARPAR-HRP conjugates, we achieved selective proximity labeling of membrane proteins in NRP-1-positive PPC1 cells, with a 3- to 5-fold increase in fluorescence intensity over controls by flow cytometry. Affinity purification and Western blotting identified a strong [~]130 kDa band corresponding to NRP-1 exclusively in labeled PPC1 cells. Mass spectrometry analysis revealed a [~]20-fold enrichment of NRP-1 and significant enrichment of integrins (ITGAV, ITGB1, ITGA3), ALCAM, EPHA2, CD109, and PLXNB2 in RPARPAR-labeled samples compared to controls. This approach could be broadly used for molecular mapping of the homing peptide interactome and its spatial proximity in live cells, streamlining the discovery of homing peptide receptors and their associated partners.

cell biology↗

bioRxiv2024

A CYCLIC PEPTIDE TARGETS GLIOBLASTOMA BY BINDING TO ABERRANTLY EXPOSED SNAP25

Disease-specific changes in tumors and other diseased tissues are an important target of research because they provide clues on the pathophysiology of the disease as well as uncovering potentially useful markers for diagnosis and treatment. Here, we report a new cyclic peptide, CESPLLSEC (CES), that specifically accumulated (homed) in intracranial U87MG and the WT-GBM model of glioblastoma from intravenous (IV) injection, associating with the vasculature. Affinity chromatography of U87MG tumor extracts on insolubilized CES peptide identified Synaptosomal Associated Protein 25 (SNAP25) as a candidate target molecule (receptor) for CES. Several results supported the identification of SNAP25 as the CES receptor. IV-injected FAM-CES colocalized with SNAP25 in the tumors, and direct binding studies showed specific CES peptide binding to recombinant human SNAP25. A CES peptide-drug conjugate designed for photodynamic therapy showed selective cytotoxicity to SNAP25+ glioblastoma cell lines. Specific accumulation of systemically injected anti-SNAP25 antibody in U87MG glioblastoma, and labeling of intact U87MG cells with anti-SNAP in flow cytometry showed that SNAP25 is available from the circulation but not in normal tissues and that it is present at the cell surface. Using an array of ECM proteins and surface plasmon resonance revealed that SNAP25 binds moderately to collagen V and strongly to collagen VI. Modeling studies suggested that CES and collagen VI compete for the same binding site on SNAP25. Our results introduce CES as a valuable targeting peptide for drug delivery, and its receptor SNAP25 as a possible molecular marker of interest for glioblastoma.

cancer biology↗

bioRxiv2024

Therapeutic Tumor Macrophage Reprogramming in Breast Cancer Through a Peptide-Drug Conjugate

In triple negative breast cancer (TNBC), pro-tumoral macrophages promote metastasis and suppress the immune response. To target these cells, we engineered a previously identified CD206 (mannose receptor)-binding peptide, mUNO, to enhance its affinity and proteolytic stability. The new rationally designed peptide, MACTIDE, includes a trypsin inhibitor loop, from the Sunflower Trypsin Inhibitor-I. Binding studies to recombinant CD206 revealed a 15-fold lower KD for MACTIDE compared to parental mUNO. Additionally, mass spectrometry showed a 5-fold increase in half-life in tumor lysate for MACTIDE compared to mUNO. Homing studies in TNBC-bearing mice showed that fluorescein (FAM)-MACTIDE precisely targeted CD206+ tumor-associated macrophages (TAMs) upon intravenous, intraperitoneal and even oral administration, with no significant accumulation in liver. We coupled MACTIDE to the FDA-approved drug Verteporfin, an established photosensitizer for photodynamic therapy and inhibitor of the YAP/TAZ pathway, to generate a conjugate here referred to as MACTIDE-V. In the orthotopic 4T1 TNBC mouse model, non-irradiated MACTIDE-V-treated mice unexpectedly showed a similar anti-tumoral effect and fewer signs of toxicity as irradiated MACTIDE-V-treated mice, leading to subsequent studies on the laser-independent activity of this conjugate. In vitro studies using bone-marrow derived mouse macrophages showed that MACTIDE-V excluded YAP from the nucleus, increased the phagocytic activity and upregulated several genes associated with cytotoxic anti-tumoral macrophages. In mouse models of TNBC, MACTIDE-V slowed primary tumor growth, suppressed lung metastases, increased markers of phagocytosis and antigen presentation in TAMs and monocytes, increasing the tumor infiltration of several lymphocyte subsets. We therefore propose MACTIDE-V as a useful peptide-drug conjugate to modulate macrophage function in the context of breast tumor immunotherapy.

cancer biology↗

bioRxiv2022

Targeting triple-negative breast cancer with β1-integrin binding aptamer

Targeted therapies have increased the treatment options for triple-negative breast cancer patients. However, the paucity of targetable biomarkers and tumour heterogeneity have limited the ability of precision-guided interventions to live up to their full potential. As affinity targeting ligands, aptamers show high selectivity towards target molecules. Compared to antibodies, aptamers have lower molecular weight, increased stability during transportation, reduced immunogenicity, and increased tissue uptake. Recently, we reported the discovery of GreenB1 aptamer that is internalized in cultured triple-negative MDA-MB-231 human breast cancer cells. We show that the GreenB1 aptamer specifically targets {beta}1-integrin, a protein previously linked to breast cancer cell invasiveness and migration. Aptamer binds to {beta}1-integrin with low nanomolar affinity. GreenB1 homes in the orthotopic 4T1 triple-negative breast cancer lesions modelled in mice. Our findings suggest potential applications for the GreenB1-guided precision agents for the diagnosis and therapy of triple-negative breast cancer.

molecular biology↗

bioRxiv2022

Paclitaxel-loaded PEGylated Cationic Lipid Nanodiscs and Small Liposomes with Protruding Brush Conformation PEG-chains Show Tumor Penetration and Proapoptotic Caspase-3 Activation

Novel approaches are required to address the urgent need to develop lipid-based carriers of paclitaxel (PTX) and other hydrophobic drugs for cancer chemotherapy. Carriers based on cationic liposomes (CLs) with fluid (i.e., chain-melted) membranes (e.g., EndoTAG-1(R)) have shown promise in preclinical and late-stage clinical studies. Recent work found that the addition of a cone-shaped poly(ethylene glycol)-lipid (PEG-lipid) to PTX-loaded CLs (CLsPTX) promotes a transition to sterically stabilized, higher-curvature (smaller) nanoparticles consisting of a mixture of PEGylated CLsPTX and PTX-containing fluid lipid nanodiscs (nanodiscsPTX). These CLsPTX and nanodiscsPTX show significantly improved uptake and cytotoxicity in cultured human cancer cells at PEG coverage in the brush regime (10 mol% PEG-lipid). Here, we studied the PTX loading, in vivo circulation half-life, and biodistribution of systemically administered CLsPTX and nanodiscsPTX and assessed their ability to induce apoptosis in triple-negative breast cancer-bearing immunocompetent mice. We focused on fluid rather than solid lipid nanodiscs because of the significantly higher solubility of PTX in fluid membranes. At 5 and 10 mol% of a PEG-lipid (PEG5K-lipid, molecular weight of PEG 5000 g/mol), the mixture of PEGylated CLsPTX and nanodiscsPTX was able to incorporate up to 2.5 mol% PTX without crystallization for at least 20 h. Remarkably, compared to preparations containing 2 and 5 mol% PEG5K-lipid (with the PEG chains in the mushroom regime), the particles at 10 mol% (with PEG chains in the brush regime) showed significantly higher blood half-life, tumor penetration and proapoptotic activity. Our study suggests that increasing the PEG coverage of CL-based drug nanoformulations can improve their pharmacokinetics and therapeutic efficacy.

biophysics↗

bioRxiv2022

ESCPE-1 Mediates Retrograde Endosomal Sorting of the SARS-CoV-2 Host Factor Neuropilin-1

Endosomal sorting maintains cellular homeostasis by recycling transmembrane proteins and associated proteins and lipids (termed cargoes) from the endosomal network to multiple subcellular destinations, including retrograde traffic to the trans-Golgi network (TGN). Viral and bacterial pathogens subvert retrograde trafficking machinery to facilitate infectivity. Here, we develop a proteomic screen to identify novel retrograde cargo proteins of the Endosomal SNX-BAR Sorting Complex Promoting Exit-1 (ESCPE-1). Using this methodology, we identify Neuropilin-1 (NRP1), a recently characterised host factor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, as a cargo directly bound and trafficked by ESCPE-1. ESCPE-1 mediates retrograde trafficking of engineered nanoparticles functionalised with the NRP1-interacting peptide of the SARS-CoV-2 Spike protein. ESCPE-1 sorting of NRP1 may therefore play a role in the intracellular membrane trafficking of NRP1-interacting viruses such as SARS-CoV-2.

cell biology↗

bioRxiv2021

Depletion of CD206+ Tumour Macrophages via a Peptide-Targeted Star-Shaped Polyglutamate Inhibits Tumourigenesis and Metastatic Dissemination in Mouse Breast Cancer Models

Although many studies have explored the depletion of tumour-associated macrophages (TAMs) as a therapeutic strategy for solid tumours, currently available compounds suffer from poor efficacy and dose-limiting side effects. Here, we developed a novel TAM-depleting agent ("OximUNO") that specifically targets CD206+ TAMs and demonstrated efficacy in triple negative breast cancer (TNBC) mouse models. OximUNO comprises a star-shaped polyglutamate (St-PGA) decorated with the CD206-targeting peptide mUNO that carries the chemotherapeutic drug doxorubicin (DOX). In TNBC models, a fluorescently labelled mUNO-decorated St-PGA homed to CD206+ TAMs within primary lesions and metastases. OximUNO exhibited no acute liver or kidney toxicity in vivo. Treatment with OximUNO reduced the progression of primary tumour lesions and pulmonary metastases, significantly diminished the number of CD206+ TAMs and increased the CD8/FOXP3 expression ratio (demonstrating immunostimulation). Our findings suggest the potential benefit of OximUNO as a TAM-depleting agent for TNBC treatment. Importantly, our studies also represent the first report of a peptide-targeted St-PGA as a targeted therapeutic nanoconjugate.

cancer biology↗

bioRxiv2021

PL1 Peptide Engages Acidic Surfaces on Tumor-Associated Fibronectin and Tenascin Isoforms to Trigger Cellular Uptake.

Tumor extracellular matrix (ECM) is a high-capacity and genetically stable target for the precision delivery of affinity ligand-guided drugs and imaging agents. Recently, we developed a PL1 peptide (sequence: PPRRGLIKLKTS) for systemic targeting of malignant ECM. Here we map the dynamics of PL1 binding to its receptors Fibronectin Extra Domain B (FN-EDB) and Tenascin C C-isoform (TNC-C) by computational modeling and cell-free binding studies on mutated receptor proteins, and study cellular binding and internalization of PL1 nanoparticles in cultured cells. Molecular dynamics simulation and docking analysis suggested that the engagement of PL1 peptide with both receptors is primarily driven by electrostatic interactions. Substituting acidic amino acid residues with neutral amino acids at predicted PL1 binding sites in FN-EDB (D52N-D49N-D12N) and TNC-C (D39N-D45N) resulted in the loss of binding of PL1 nanoparticles. Remarkably, PL1-functionalized nanoparticles (NPs) were not only deposited on the target ECM but bound the cells and initiated a robust cellular uptake via a pathway resembling macropinocytosis. Our studies establish the mode of engagement of the PL1 peptide with its receptors and suggest applications for intracellular delivery of nanoscale payloads. The outcomes of this work can be used for the development of PL1-derived peptides with improved stability, affinity and specificity for precision targeting of the tumor ECM and malignant cells. One Sentence SummaryPL1 peptide is recruited to the acidic surfaces on oncofetal fibronectin EDB and tenascin C-C isoform, triggering cellular uptake of PL1-functionalized nanoparticles.

cancer biology↗

bioRxiv2020

In vivo phage display: identification of organ-specific peptides using deep sequencing and differential profiling across tissues

In vivo phage display is widely used for identification of organ- or disease-specific homing peptides. However, the current in vivo phage biopanning approaches fail to assess biodistribution of specific peptide phages across tissues during the screen, thus necessitating laborious and time-consuming post-screening validation studies on individual peptide phages. Here, we adopted bioinformatics tools used for RNA sequencing for analysis of high throughput sequencing (HTS) data to estimate the representation of individual peptides during biopanning in vivo. The data from in vivo phage screen were analyzed using differential binding - relative representation of each peptide in the target organ vs. in a panel of control organs. Application of this approach in a model study using low-diversity peptide T7 phage library with spiked-in brain homing phage, demonstrated brain-specific differential binding of brain homing phage and resulted in identification of novel lung and brain specific homing peptides. Our study provides a broadly applicable approach to streamline in vivo peptide phage biopanning and to increase its reproducibility and success rate. Graphic abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=90 SRC="FIGDIR/small/181974v2_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@1853e75org.highwire.dtl.DTLVardef@c7ad8corg.highwire.dtl.DTLVardef@152f4feorg.highwire.dtl.DTLVardef@15261d0_HPS_FORMAT_FIGEXP M_FIG C_FIG In vivo phage display using differential binding approach

molecular biology↗

bioRxiv2020

Neuropilin-1 facilitates SARS-CoV-2 cell entry and provides a possible pathway into the central nervous system

The causative agent of the current pandemic and coronavirus disease 2019 (COVID-19) is the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)1. Understanding how SARS-CoV-2 enters and spreads within human organs is crucial for developing strategies to prevent viral dissemination. For many viruses, tissue tropism is determined by the availability of virus receptors on the surface of host cells2. Both SARS-CoV and SARS-CoV-2 use angiotensin-converting enzyme 2 (ACE2) as a host receptor, yet, their tropisms differ3-5. Here, we found that the cellular receptor neuropilin-1 (NRP1), known to bind furin-cleaved substrates, significantly potentiates SARS-CoV-2 infectivity, which was inhibited by a monoclonal blocking antibody against the extracellular b1b2 domain of NRP1. NRP1 is abundantly expressed in the respiratory and olfactory epithelium, with highest expression in endothelial cells and in the epithelial cells facing the nasal cavity. Neuropathological analysis of human COVID-19 autopsies revealed SARS-CoV-2 infected NRP1-positive cells in the olfactory epithelium and bulb. In the olfactory bulb infection was detected particularly within NRP1-positive endothelial cells of small capillaries and medium-sized vessels. Studies in mice demonstrated, after intranasal application, NRP1-mediated transport of virus-sized particles into the central nervous system. Thus, NRP1 could explain the enhanced tropism and spreading of SARS-CoV-2.

microbiology↗

bioRxiv2020

Neuropilin-1 is a host factor for SARS-CoV-2 infection

SARS-CoV-2 is the causative agent of COVID-19, a coronavirus disease that has infected more than 6.6 million people and caused over 390,000 deaths worldwide1,2. The Spike (S) protein of the virus forms projections on the virion surface responsible for host cell attachment and penetration. This viral glycoprotein is synthesized as a precursor in infected cells and, to be active, must be cleaved to two associated polypeptides: S1 and S2(3,4). For SARS-CoV-2 the cleavage is catalysed by furin, a host cell protease, which cleaves the S protein precursor at a specific sequence motif that generates a polybasic Arg-Arg-Ala-Arg (RRAR) C-terminal sequence on S1. This sequence motif conforms to the C-end rule (CendR), which means that the C-terminal sequence may allow the protein to associate with cell surface neuropilin-1 (NRP1) and neuropilin-2 (NRP2) receptors5. Here we demonstrate using immunoprecipitation, site-specific mutagenesis, structural modelling, and antibody blockade that, in addition to engaging the known receptor ACE2, S1 can bind to NRP1 through the canonical CendR mechanism. This interaction enhances infection by SARS-CoV-2 in cell culture. NRP1 thus serves as a host factor for SARS-CoV-2 infection, and provides a therapeutic target for COVID-19.

cell biology↗

bioRxiv2017

Detection of small breast tumors using tumor penetrating-polymersomes engineered to target p32 protein

Triple negative breast cancer (TNBC) is the deadliest form of breast cancer and its successful treatment critically depends on early diagnosis and therapy. The multi-compartment protein p32 is overexpressed and present at cell surfaces in a variety of tumors, including TNBC, specifically in the malignant cells and endothelial cells, and in macrophages localized in hypoxic areas of the tumor. Herein we used polyethylene glycol-polycaprolactone polymersomes that were affinity targeted with the p32-binding tumor penetrating peptide LinTT1 (AKRGARSTA) for imaging of TNBC lesions. A tyrosine residue was added to the peptide to allow for 124I labeling and PET imaging. In a TNBC model in mice, systemic LinTT1-targeted polymersomes accumulated in early tumor lesions more than twice as efficiently as untargeted polymersomes with up to 20% ID/cc at 24 h after administration. The PET-imaging was very sensitive, allowing detection of tumors as small as [~]20mm3. Confocal imaging of tumor tissue sections revealed a high degree of vascular exit and stromal penetration of LinTT1-targeted polymersomes and co-localization with tumor-associated macrophages. Our studies show that systemic LinTT1-targeted polymersomes can be potentially used for precision-guided tumor imaging and treatment of TNBC.

cancer biology↗

bioRxiv2017

Competition of Charge-Mediated and Specific Binding by Peptide-Tagged Cationic Liposome-DNA Nanoparticles In Vitro and In Vivo

Cationic liposome-nucleic acid (CL-NA) complexes, which form spontaneously, are a highly modular gene delivery system. These complexes can be sterically stabilized via PEGylation [PEG: poly(ethylene glycol)] into nanoparticles (NPs) and targeted to specific tissues and cell types via the conjugation of an affinity ligand. However, there are currently no guidelines on how to effectively navigate the large space of compositional parameters that modulate the specific and nonspecific binding interactions of peptide-targeted NPs with cells. Such guidelines are desirable to accelerate the optimization of formulations with novel peptides. Using PEG-lipids functionalized with a library of prototypical tumor-homing peptides, we varied the peptide density and other parameters (binding motif, peptide charge, CL/DNA charge ratio) to study their effect on the binding and uptake of the corresponding NPs. We used flow cytometry to quantitatively assess binding as well as internalization of NPs by cultured cancer cells. Surprisingly, full peptide coverage resulted in less binding and internalization than intermediate coverage, with the optimum coverage varying between cell lines. In, addition, our data revealed that great care must be taken to prevent nonspecific electrostatic interactions from interfering with the desired specific binding and internalization. Importantly, such considerations must take into account the charge of the peptide ligand as well as the membrane charge density and the CL/DNA charge ratio. To test our guidelines, we evaluated the in vivo tumor selectivity of selected NP formulations in a mouse model of peritoneally disseminated human gastric cancer. Intraperitoneally administered peptide-tagged CL-DNA NPs showed tumor binding, minimal accumulation in healthy control tissues, and preferential penetration of smaller tumor nodules, a highly clinically relevant target known to drive recurrence of the peritoneal cancer.\n\nGraphical Abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

biophysics↗