bims-cepepe 2024-03-24 papers

bims-cepepe

Biomed News

on Cell-penetrating peptides

Issue of 2024‒03‒24
twenty-six papers selected by
Henry Lamb, Queensland University of Technology

Peptide-encoding gene transfer to modulate intracellular protein-protein interactions.
Identification and Development of Cyclic Peptide Inhibitors of Hypoxia Inducible Factors 1 and 2 That Disrupt Hypoxia-Response Signaling in Cancer Cells.
Identification of "Structural Pin" Interactions and Their Significance for the Conformational Control of Macrocyclic Scaffolds.
In silico design of potential Mcl-1 peptide-based inhibitors.
Peptide-derived ligands for the discovery of safer opioid analgesics.
Enhancement of Oral Bioavailability of Protein and Peptide by Polysaccharide-based Nanoparticles.
Second generation Al18F-labeled D-amino acid peptide for CXCR4 targeted molecular imaging.
An Overview of Peptides and Peptide Pools for Antigen-Specific Stimulation in T-Cell Assays.
Neutrophil elastase as a versatile cleavage enzyme for activation of αvβ3 integrin-targeted small molecule drug conjugates with different payload classes in the tumor microenvironment.
Folding of class IIa HDAC derived peptides into α-helices upon binding to myocyte enhancer factor-2 in complex with DNA.
Small Fluorogenic Amino Acids for Peptide-Guided Background-Free Imaging.
Computational design of soluble functional analogues of integral membrane proteins.
Nanoparticle-Based Combinational Strategies for Overcoming the Blood-Brain Barrier and Blood-Tumor Barrier.
Targeted intracellular delivery of dimeric STINGa by two pHLIP peptides for treatment of solid tumors.
Post-translational modifications of beta-amyloid alter its transport in the blood-brain barrier in vitro model.
ANN multi-layer perceptron for prediction of blood-brain barrier permeable compounds for central nervous system therapeutics.
High-Throughput Computational Screening of Solid-Binding Peptides.
Mutational scanning pinpoints distinct binding sites of key ATGL regulators in lipolysis.
Multielectrode array characterization of human induced pluripotent stem cell derived neurons in co-culture with primary human astrocytes.
Enzyme-Activatable Chemokine Conjugates for In Vivo Targeting of Tumor-Associated Macrophages.
The Complementarity of Nuclear Magnetic Resonance and Native Mass Spectrometry in Probing Protein-Protein Interactions.
The SecM arrest peptide traps a pre-peptide bond formation state of the ribosome.
Current approaches in glioblastoma multiforme immunotherapy.
Identification of Novel Series of Potent and Selective Relaxin Family Peptide Receptor 1 (RXFP1) Agonists.
Long-Residence Time Peptide Antagonist for the Vasopressin V2 Receptor to Treat Autosomal Dominant Polycystic Kidney Disease.
Impact of Resistance on Therapeutic Design: A Moran Model of Cancer Growth.

Mol Ther Methods Clin Dev. 2024 Jun 13. 32(2): 101226

Peptide-encoding gene transfer to modulate intracellular protein-protein interactions.

Toshihiko Taya, Daisuke Kami, Fumiya Teruyama, Satoaki Matoba, Satoshi Gojo.

  Peptide drug discovery has great potential, but the cell membrane is a major obstacle when the target is an intracellular protein-protein interaction (PPI). It is difficult to target PPIs with small molecules; indeed, there are no intervention tools that can target any intracellular PPI. In this study, we developed a platform that enables the introduction of peptides into cells via mRNA-based gene delivery. Peptide-length nucleic acids do not enable stable ribosome binding and exhibit little to no translation into protein. In this study, a construct was created in which the sequence encoding dihydrofolate reductase (DHFR) was placed in front of the sequence encoding the target peptide, together with a translation skipping sequence, as a sequence that meets the requirements of promoting ribosome binding and rapid decay of the translated protein. This enabled efficient translation from the mRNA encoding the target protein while preventing unnecessary protein residues. Using this construct, we showed that it can inhibit Drp1/Fis1 binding, one of the intracellular PPIs, which governs mitochondrial fission, an important aspect of mitochondrial dynamics. In addition, it was shown to inhibit pathological hyperfission, normalize mitochondrial dynamics and metabolism, and inhibit apoptosis of the mitochondrial pathway.

Keywords:  destabilizing domain; gene transfer; mRNA medicine; peptide; ribosome

DOI:  https://doi.org/10.1016/j.omtm.2024.101226
J Am Chem Soc. 2024 Mar 19.

Identification and Development of Cyclic Peptide Inhibitors of Hypoxia Inducible Factors 1 and 2 That Disrupt Hypoxia-Response Signaling in Cancer Cells.

Andrew T Ball, Soran Mohammed, Cyrielle Doigneaux, Reece M Gardner, James W Easton, Steven Turner, Jonathan W Essex, Garry Pairaudeau, Ali Tavassoli.

Hypoxia inducible factor (HIF) is a heterodimeric transcription factor composed of an oxygen-regulated α subunit and a constitutively expressed β subunit that serves as the master regulator of the cellular response to low oxygen concentrations. The HIF transcription factor senses and responds to hypoxia by significantly altering transcription and reprogramming cells to enable adaptation to a hypoxic microenvironment. Given the central role played by HIF in the survival and growth of tumors in hypoxia, inhibition of this transcription factor serves as a potential therapeutic approach for treating a variety of cancers. Here, we report the identification, optimization, and characterization of a series of cyclic peptides that disrupt the function of HIF-1 and HIF-2 transcription factors by inhibiting the interaction of both HIF-1α and HIF-2α with HIF-1β. These compounds are shown to bind to HIF-α and disrupt the protein-protein interaction between the α and β subunits of the transcription factor, resulting in disruption of hypoxia-response signaling by our lead molecule in several cancer cell lines.

DOI: https://doi.org/10.1021/jacs.3c10508
Angew Chem Int Ed Engl. 2024 Mar 18. e202402372

Identification of "Structural Pin" Interactions and Their Significance for the Conformational Control of Macrocyclic Scaffolds.

Solomon D Appavoo, Nicholas W Heller, Christiaan T van Campenhout, George J Saunders, Andrei K Yudin.

  While peptide macrocycles with rigid conformations have proven to be useful in the design of chemical probes of protein targets, conformational flexibility and rapid interconversion can be equally vital for biological activity and favorable physicochemical properties. This study introduces the concept of "structural pin", which describes a hydrogen bond that is largely responsible for stabilizing the entire macrocycle backbone conformation. Structural analysis of macrocycles using nuclear magnetic resonance (NMR), molecular modelling and X-ray diffraction indicates that disruption of the structural pin can drastically influence the conformation of the entire ring, resulting in novel states with increased flexibility. This finding provides a new tool to interrogate dynamic behaviour of macrocycles. Identification of structural pins offers a potentially useful conceptual framework to understand positions that can either be modified to give flexible structures or retained to maintain the rigidity of the scaffold.

Keywords:  cyclic peptides, conformational analysis, macrocycles, flexibility, chameleonicity

DOI:  https://doi.org/10.1002/anie.202402372
J Mol Model. 2024 Mar 18. 30(4): 108

In silico design of potential Mcl-1 peptide-based inhibitors.

Naser Faraji, Norelle L Daly, Seyed Shahriar Arab, Ahmad Yari Khosroushahi.

  CONTEXT: BIM (Bcl-2 interacting mediator of apoptosis)-derived peptides that specifically target over-expressed Mcl-1 (myeloid cell leukemia-1) protein and induce apoptosis are potentially anti-cancer agents. Since the helicity of BIM-derived peptides has a crucial role in their functionality, a range of strategies have been used to increase the helicity including the introduction of unnatural residues and stapling methods that have some drawbacks such as the accumulation in the liver. To avoid these drawbacks, this study aimed to design a more helical peptide by utilizing bioinformatics algorithms and molecular dynamics simulations without exploiting unnatural residues and stapling methods. MM-PBSA results showed that the mutations of A4fE and A2eE in analogue 5 demonstrate a preference towards binding with Mcl-1. As evidenced by Circular dichroism results, the helicity increases from 18 to 34%, these findings could enhance the potential of analogue 5 as an anti-cancer agent targeting Mcl-1. The applied strategies in this research could shed light on the in silico peptide design. Moreover, analogue 5 as a drug candidate can be evaluated in vitro and in vivo studies.METHODS: The sequence of the lead peptide was determined using the ApInAPDB database and PRALINE program. Contact finder and PDBsum web server softwares were used to determine the contact involved amino acids in complex with Mcl-1. All identified salt bridge contributing residues were unaltered to preserve the binding affinity. After proposing novel analogues, their secondary structures were predicted by Cham finder web server software and GOR, Neural Network, and Chou-Fasman algorithms. Finally, molecular dynamics simulations run for 100 ns were done using the GROMACS, version 5.0.7, with the CHARMM36 force field. MM-PBSA was used to assess binding affinity specificity in targeting Mcl-1 and Bcl-xL (B-cell lymphoma extra-large).

Keywords:  Apoptosis; BIM peptide; Mcl-1; Molecular dynamic simulation; Peptide design

DOI:  https://doi.org/10.1007/s00894-024-05901-8
Drug Discov Today. 2024 Mar 19. pii: S1359-6446(24)00075-8. [Epub ahead of print] 103950

Peptide-derived ligands for the discovery of safer opioid analgesics.

Abbe Eliasof, Lee-Yuan Liu-Chen, Yangmei Li.

  Drugs targeting the μ-opioid receptor (MOR) remain the most efficacious analgesics for the treatment of pain, but activation of MOR with current opioid analgesics also produces harmful side effects, notably physical dependence, addiction, and respiratory depression. Opioid peptides have been accepted as promising candidates for the development of safer and more efficacious analgesics. To develop peptide-based opioid analgesics, strategies such as modification of endogenous opioid peptides, development of multifunctional opioid peptides, G protein-biased opioid peptides, and peripherally restricted opioid peptides have been reported. This review seeks to provide an overview of the opioid peptides that produce potent antinociception with much reduced side effects in animal models and highlight the potential advantages of peptides as safer opioid analgesics. Teaser: Peptide-based opioid ligands are promising candidates for the discovery and development of efficacious, safer, and non-/less addictive analgesics.

Keywords:  opioid receptor ligand; pain; peptide; safe analgesic; structural modification

DOI:  https://doi.org/10.1016/j.drudis.2024.103950
Protein Pept Lett. 2024 Mar 20.

Enhancement of Oral Bioavailability of Protein and Peptide by Polysaccharide-based Nanoparticles.

Md Moidul Islam, Sarjana Raikwar.

  Oral drug delivery is a prevalent and cost-effective method due to its advantages, such as increased drug absorption surface area and improved patient compliance. However, delivering proteins and peptides orally remains a challenge due to their vulnerability to degradation by digestive enzymes, stomach acids, and limited intestinal membrane permeability, resulting in poor bioavailability. The use of nanotechnology has emerged as a promising solution to enhance the bioavailability of these vital therapeutic agents. Polymeric NPs, made from natural or synthetic polymers, are commonly used. Natural polysaccharides, such as alginate, chitosan, dextran, starch, pectin, etc., have gained preference due to their biodegradability, biocompatibility, and versatility in encapsulating various drug types. Their hydrophobic-hydrophilic properties can be tailored to suit different drug molecules.

Keywords:  Oral bioavailability; Peptides; Polysaccharide-based nanoparticles; Proteins; drug delivery

DOI:  https://doi.org/10.2174/0109298665292469240228064739
Nucl Med Biol. 2024 Mar 19. pii: S0969-8051(24)00032-5. [Epub ahead of print]132-133 108906

Second generation Al18F-labeled D-amino acid peptide for CXCR4 targeted molecular imaging.

Muriel Aline Spahn, Kaat Luyten, Tom Van Loy, Mike Sathekge, Christophe M Deroose, Michel Koole, Dominique Schols, Wim Vanduffel, Kristof De Vos, Pieter Annaert, Guy Bormans, Frederik Cleeren.

  BACKGROUND: The C-X-C chemokine receptor type 4 (CXCR4) is overexpressed in many cancers, e.g. multiple myeloma and acute leukemia, yet solely [68Ga]PentixaFor is used for clinical PET imaging. The aim of this study was to develop and assess a second generation Al18F-labeled D-amino acid peptide based on the viral macrophage inflammatory protein II for CXCR4 targeted molecular imaging.METHODS: We designed a library of monomer and multimer constructs and evaluated their binding affinity for human and mouse CXCR4. Based on these results, we selected the best vector molecule for development of an Al18F-labeled ligand, [18F]AlF-NOTA-2xDV1(c11sc12s), which was further evaluated in a cell-based binding assay to assess its binding properties and specificity for CXCR4. Next, pharmacokinetics and tumor uptake of [18F]AlF-NOTA-2xDV1(c11sc12s) were evaluated in naïve mice and mice with xenografts derived from U87.CXCR4 cells. Finally, we performed an imaging study in a non-human primate to assess the in vivo distribution of this novel radioligand in a species closely related to humans.
RESULTS: The lead ligand AlF-NOTA-2xDV1(c11sc12s) showed six-fold higher affinity for human CXCR4 compared to Ga-Pentixafor. The corresponding radiotracer was obtained in a good radiochemical yield of 40.1 ± 13.5 % (n = 4) and apparent molar activity of 20.4 ± 3.3 MBq/nmol (n = 4) after optimization. In U87.CD4.CXCR4 cell binding assays, the total bound fraction of [18F]AlF-NOTA-(2×)DV1(c11sc12s) was 32.4 ± 1.8 %. This fraction could be reduced by 82.5 % in the presence of 75 μM AMD3100. In naïve mice, [18F]AlF-NOTA-2xDV1(c11sc12s) accumulated in organs expressing mouse CXCR4, e.g. the liver (SUVmean (mean standardized uptake value) 75 min p.i. 11.7 ± 0.6), which was blockable by co-injecting AMD3100 (5 mg/kg). In U87.CXCR4 xenografted tumor mice, the tumor uptake of [18F]AlF-NOTA-2xDV1(c11sc12s) remained low (SUVmean 0.5 ± 0.1), but was reduced by co-administration of AMD3100. Surprisingly, [18F]AlF-NOTA-2xDV1(c11sc12s) exhibited a similar biodistribution in a non-human primate as in mice indicating off-target binding of [18F]AlF-NOTA-2xDV1(c11sc12s) in liver tissue. We confirmed that [18F]AlF-NOTA-2xDV1(c11sc12s) is taken up by hepatocytes using in vitro studies and that the uptake can be blocked with AMD3100 and rifampicin, a potent organic anion-transporting-polypeptide (OATP)1B1 and OATP1B3 inhibitor.
CONCLUSION: The second generation D-peptide AlF-NOTA-2xDV1(c11sc12s) showed high affinity for human CXCR4 and the corresponding radiotracer was produced in good radiochemical yields. However, [18F]AlF-NOTA-2xDV1(c11sc12s) is not specific for CXCR4 and is also a substrate for OATP1B1 and/or OATP1B3, known to mediate hepatic uptake. Therefore, D-amino acid peptides, based on the viral macrophage inflammatory protein II, are not the prefered vector molecule for the development of CXCR4 targeting molecular imaging tools.

Keywords:  Al18F; CXCR4; D-peptide; Positron emission tomography; Radiotracer

DOI:  https://doi.org/10.1016/j.nucmedbio.2024.108906
Methods Mol Biol. 2024 ;2768 29-50

An Overview of Peptides and Peptide Pools for Antigen-Specific Stimulation in T-Cell Assays.

Karsten Schnatbaum, Pavlo Holenya, Sebastian Pfeil, Michael Drosch, Maren Eckey, Ulf Reimer, Holger Wenschuh, Florian Kern.

  The analysis of antigen-specific T-cell responses has become routine in many laboratories. Functional T-cell assays like enzyme-linked-immuno-spot (ELISPOT), which depend on antigen-specific stimulation, increasingly use peptides to represent the antigen of interest. Besides single peptides, mixtures of peptides (peptide pools) are very frequently applied. Such peptide pools may, for example, represent entire proteins (with overlapping peptides covering a protein sequence) or include noncontiguous peptides such as a collection of T-cell-stimulating peptides. The optimum specification of single peptides or peptide pools for T-cell stimulation assays will depend on the purpose of the test, the target T-cell population, the availability of sample, requirements regarding reproducibility, and, last but not least, the available budget, to mention only the most important factors. Because of the way peptides are produced, they will always contain certain amounts of impurities such as peptides with deletions or truncated peptides, and there may be additional by-products of peptide synthesis. Optimized synthesis protocols as well as purification help reduce impurities that might otherwise cause false-positive assay results. However, specific requirements with respect to purity will vary depending on the purpose of an assay. Finally, storage conditions significantly affect the shelf life of peptides, which is relevant especially for longitudinal studies. The present book chapter addresses all of these aspects in detail. It should provide the researcher with all necessary background knowledge for making the right decisions when it comes to choosing, using, and storing peptides for ELISPOT and other T-cell stimulation assays.

Keywords:  Capping; Deletion peptides; ELISPOT; Epitope mapping; Peptide libraries; Peptide pools; Peptide purity; Peptide solvents; Peptide storage; Peptides; Protein-spanning peptide pools; Solid phase peptide synthesis; T-cell activation assays

DOI:  https://doi.org/10.1007/978-1-0716-3690-9_3
Front Pharmacol. 2024 ;15 1358393

Neutrophil elastase as a versatile cleavage enzyme for activation of αvβ3 integrin-targeted small molecule drug conjugates with different payload classes in the tumor microenvironment.

Anne-Sophie Rebstock, Mareike Wiedmann, Beatrix Stelte-Ludwig, Harvey Wong, Amy J Johnson, Raquel Izumi, Ahmed Hamdy, Hans-Georg Lerchen.

  Introduction: The development of bioconjugates for the targeted delivery of anticancer agents is gaining momentum after recent success of antibody drug conjugates (ADCs) in the clinic. Smaller format conjugates may have several advantages including better tumor penetration; however, cellular uptake and trafficking may be substantially different from ADCs. To fully leverage the potential of small molecule drug conjugates (SMDCs) with potent binding molecules mediating tumor homing, novel linker chemistries susceptible for efficient extracellular activation and payload release in the tumor microenvironment (TME) need to be explored. Methods: We designed a novel class of SMDCs, which target αvβ3 integrins for tumor homing and are cleaved by neutrophil elastase (NE), a serine protease active in the TME. A peptidomimetic αvβ3 ligand was attached via optimized linkers composed of substrate peptide sequences of NE connected to different functional groups of various payload classes, such as camptothecins, monomethyl auristatin E, kinesin spindle protein inhibitors (KSPi) and cyclin-dependent kinase 9 inhibitors (CDK-9i). Results: NE-mediated cleavage was found compatible with the diverse linker attachments via hindered ester bonds, amide bonds and sulfoximide bonds. Efficient and traceless release of the respective payloads was demonstrated in biochemical assays. The newly designed SMDCs were highly stable in buffer as well as in rat and human plasma. Cytotoxicity of the SMDCs in cancer cell lines was clearly dependent on NE. IC50 values were in the nanomolar or sub-nanomolar range across several cancer cell lines reaching similar potencies as compared to the respective payloads only in the presence of NE. In vivo pharmacokinetics evaluating SMDC and free payload exposures in rat and particularly the robust efficacy with good tolerability in triple negative breast and small cell lung cancer murine models demonstrate the utility of this approach for selective delivery of payloads to the tumor. Discussion: These results highlight the broad scope of potential payloads and suitable conjugation chemistries paving the way for future SMDCs harnessing the safety features of targeted delivery approaches in combination with NE cleavage in the TME.

Keywords:  camptothecin; neutrophil elastase (NE); small molecule drug conjugate; tumor microenvironment; αvβ3 integrin

DOI:  https://doi.org/10.3389/fphar.2024.1358393
J Mol Biol. 2024 Mar 14. pii: S0022-2836(24)00136-0. [Epub ahead of print] 168541

Folding of class IIa HDAC derived peptides into α-helices upon binding to myocyte enhancer factor-2 in complex with DNA.

Monica Chinellato, Stefano Perin, Alberto Carli, Luana Lastella, Barbara Biondi, Giuseppe Borsato, Eros Di Giorgio, Claudio Brancolini, Laura Cendron, Alessandro Angelini.

  Interaction of transcription factor myocyte enhancer factor-2 (MEF2) family members with class IIa histone deacetylases (HDACs) has been implicated in a wide variety of diseases. Though considerable knowledge on this topic has been accumulated over the years, a high resolution and detailed analysis of the binding mode of multiple class IIa HDAC derived peptides with MEF2D is still lacking. To fulfil this gap, we report here the crystal structure of MEF2D in complex with double strand DNA and four different class IIa HDAC derived peptides, namely HDAC4, HDAC5, HDAC7 and HDAC9. All class IIa HDAC derived peptides form extended amphipathic α-helix structures that fit snugly in the hydrophobic groove of MEF2D domain. Binding mode of class IIa HDAC derived peptides to MEF2D is very similar and occur primarily through nonpolar interactions mediated by highly conserved branched hydrophobic amino acids. Further studies revealed that class IIa HDAC derived peptides are unstructured in solution and appear to adopt a folded α-helix structure only upon binding to MEF2D. Comparison of our peptide-protein complexes with previously characterized structures of MEF2 bound to different co-activators and co-repressors, highlighted both differences and similarities, and revealed the adaptability of MEF2 in protein-protein interactions. The elucidation of the three-dimensional structure of MEF2D in complex with multiple class IIa HDAC derived peptides provide not only a better understanding of the molecular basis of their interactions but also have implications for the development of novel antagonist.

Keywords:  DNA-binding; HDAC; MEF2; class IIa histone deacetylases; folding-upon-binding; myocyte enhancer factor-2; peptide; protein-protein interaction; transcription factor

DOI:  https://doi.org/10.1016/j.jmb.2024.168541
Angew Chem Weinheim Bergstr Ger. 2023 Jan 23. 135(4): e202216231

Small Fluorogenic Amino Acids for Peptide-Guided Background-Free Imaging.

Fabio de Moliner, Zuzanna Konieczna, Lorena Mendive-Tapia, Rebecca S Saleeb, Katie Morris, Juan Antonio Gonzalez-Vera, Takeshi Kaizuka, Seth G N Grant, Mathew H Horrocks, Marc Vendrell.

  The multiple applications of super-resolution microscopy have prompted the need for minimally invasive labeling strategies for peptide-guided fluorescence imaging. Many fluorescent reporters display limitations (e.g., large and charged scaffolds, non-specific binding) as building blocks for the construction of fluorogenic peptides. Herein we have built a library of benzodiazole amino acids and systematically examined them as reporters for background-free fluorescence microscopy. We have identified amine-derivatized benzoselenadiazoles as scalable and photostable amino acids for the straightforward solid-phase synthesis of fluorescent peptides. Benzodiazole amino acids retain the binding capabilities of bioactive peptides and display excellent signal-to-background ratios. Furthermore, we have demonstrated their application in peptide-PAINT imaging of postsynaptic density protein-95 nanoclusters in the synaptosomes from mouse brain tissues.

Keywords:  Fluorescence; Microscopy; Probes; Proteins; Super-Resolution

DOI:  https://doi.org/10.1002/ange.202216231
bioRxiv. 2024 Mar 07. pii: 2023.05.09.540044. [Epub ahead of print]

Computational design of soluble functional analogues of integral membrane proteins.

Casper A Goverde, Martin Pacesa, Nicolas Goldbach, Lars J Dornfeld, Petra E M Balbi, Sandrine Georgeon, Stéphane Rosset, Srajan Kapoor, Jagrity Choudhury, Justas Dauparas, Christian Schellhaas, Simon Kozlov, David Baker, Sergey Ovchinnikov, Alex J Vecchio, Bruno E Correia.

De novo design of complex protein folds using solely computational means remains a significant challenge. Here, we use a robust deep learning pipeline to design complex folds and soluble analogues of integral membrane proteins. Unique membrane topologies, such as those from GPCRs, are not found in the soluble proteome and we demonstrate that their structural features can be recapitulated in solution. Biophysical analyses reveal high thermal stability of the designs and experimental structures show remarkable design accuracy. The soluble analogues were functionalized with native structural motifs, standing as a proof-of-concept for bringing membrane protein functions to the soluble proteome, potentially enabling new approaches in drug discovery. In summary, we designed complex protein topologies and enriched them with functionalities from membrane proteins, with high experimental success rates, leading to a de facto expansion of the functional soluble fold space.

DOI: https://doi.org/10.1101/2023.05.09.540044
Int J Nanomedicine. 2024 ;19 2529-2552

Nanoparticle-Based Combinational Strategies for Overcoming the Blood-Brain Barrier and Blood-Tumor Barrier.

Su Hyun Lim, Gi Taek Yee, Dongwoo Khang.

  The blood-brain barrier (BBB) and blood-tumor barrier (BTB) pose substantial challenges to efficacious drug delivery for glioblastoma multiforme (GBM), a primary brain tumor with poor prognosis. Nanoparticle-based combinational strategies have emerged as promising modalities to overcome these barriers and enhance drug penetration into the brain parenchyma. This review discusses various nanoparticle-based combinatorial approaches that combine nanoparticles with cell-based drug delivery, viral drug delivery, focused ultrasound, magnetic field, and intranasal drug delivery to enhance drug permeability across the BBB and BTB. Cell-based drug delivery involves using engineered cells as carriers for nanoparticles, taking advantage of their intrinsic migratory and homing capabilities to facilitate the transport of therapeutic payloads across BBB and BTB. Viral drug delivery uses engineered viral vectors to deliver therapeutic genes or payloads to specific cells within the GBM microenvironment. Focused ultrasound, coupled with microbubbles or nanoparticles, can temporarily disrupt the BBB to increase drug permeability. Magnetic field-guided drug delivery exploits magnetic nanoparticles to facilitate targeted drug delivery under an external magnetic field. Intranasal drug delivery offers a minimally invasive avenue to bypass the BBB and deliver therapeutic agents directly to the brain via olfactory and trigeminal pathways. By combining these strategies, synergistic effects can enhance drug delivery efficiency, improve therapeutic efficacy, and reduce off-target effects. Future research should focus on optimizing nanoparticle design, exploring new combination strategies, and advancing preclinical and clinical investigations to promote the translation of nanoparticle-based combination therapies for GBM.

Keywords:  blood-brain barrier; blood-tumor barrier; combination strategy; glioblastoma; intranasal drug delivery; magnetic field; nanoparticle; ultrasound-wave

DOI:  https://doi.org/10.2147/IJN.S450853
Front Pharmacol. 2024 ;15 1346756

Targeted intracellular delivery of dimeric STINGa by two pHLIP peptides for treatment of solid tumors.

Anna Moshnikova, Michael DuPont, Marissa Iraca, Craig Klumpp, Hannah Visca, Dana Allababidi, Phoebe Pelzer, Donald M Engelman, Oleg A Andreev, Yana K Reshetnyak.

  Introduction: We have developed a delivery approach that uses two pHLIP peptides that collaborate in the targeted intracellular delivery of a single payload, dimeric STINGa (dMSA). Methods: dMSA was conjugated with two pHLIP peptides via S-S cleavable self-immolating linkers to form 2pHLIP-dMSA. Results: Biophysical studies were carried out to confirm pH-triggered interactions of the 2pHLIP-dMSA with membrane lipid bilayers. The kinetics of linker self-immolation and dMSA release, the pharmacokinetics, the binding to plasma proteins, the stability of the agent in plasma, the targeting and resulting cytokine activation in tumors, and the biodistribution of the construct was investigated. This is the first study demonstrating that combining the energy of the membrane-associated folding of two pHLIPs can be utilized to enhance the targeted intracellular delivery of large therapeutic cargo payloads. Discussion: Linking two pHLIPs to the cargo extends blood half-life, and targeted delivery of dimeric STINGa induces tumor eradication and the development of robust anti-cancer immunity.

Keywords:  biophysics; cold tumors; imaging; immuno-suppressive tumors; tumor acidity

DOI:  https://doi.org/10.3389/fphar.2024.1346756
Front Mol Neurosci. 2024 ;17 1362581

Post-translational modifications of beta-amyloid alter its transport in the blood-brain barrier in vitro model.

Kseniya B Varshavskaya, Irina Yu Petrushanko, Vladimir A Mitkevich, Evgeny P Barykin, Alexander A Makarov.

  One of the hallmarks of Alzheimer's disease (AD) is the accumulation of beta-amyloid peptide (Aβ) leading to formation of soluble neurotoxic Aβ oligomers and insoluble amyloid plaques in various parts of the brain. Aβ undergoes post-translational modifications that alter its pathogenic properties. Aβ is produced not only in brain, but also in the peripheral tissues. Such Aβ, including its post-translationally modified forms, can enter the brain from circulation by binding to RAGE and contribute to the pathology of AD. However, the transport of modified forms of Aβ across the blood-brain barrier (BBB) has not been investigated. Here, we used a transwell BBB model as a controlled environment for permeability studies. We found that Aβ42 containing isomerized Asp7 residue (iso-Aβ42) and Aβ42 containing phosphorylated Ser8 residue (pS8-Aβ42) crossed the BBB better than unmodified Aβ42, which correlated with different contribution of endocytosis mechanisms to the transport of these isoforms. Using microscale thermophoresis, we observed that RAGE binds to iso-Aβ42 an order of magnitude weaker than to Aβ42. Thus, post-translational modifications of Aβ increase the rate of its transport across the BBB and modify the mechanisms of the transport, which may be important for AD pathology and treatment.

Keywords:  Alzheimer's disease; beta-amyloid; blood-brain barrier; caveolin-dependent endocytosis; clathrin-dependent endocytosis; post-translational modifications; rage

DOI:  https://doi.org/10.3389/fnmol.2024.1362581
J Biomol Struct Dyn. 2024 Mar 18. 1-6

ANN multi-layer perceptron for prediction of blood-brain barrier permeable compounds for central nervous system therapeutics.

Aditi Sharma, Subathra Selvam, Priya Dharshini Balaji, Thirumurthy Madhavan.

  Endothelial cells produce a semipermeable barrier known as the blood-brain barrier (BBB) to keep undesired chemicals out of the central nervous system (CNS). However, this barrier also restricts the exploration of potential new medications due to insufficient exposure. To address this challenge, machine learning (ML) algorithms can be useful to predict the BBB permeability of chemical compounds. Support vector machines, continuous neural networks, and deep learning approaches have been used to identify compounds that can penetrate the BBB. However, predicting BBB permeability based solely on chemical structure can be difficult. In the current research, we developed an ML model using a large dataset to predict BBB permeability, which could be used for early-stage drug screening of potential CNS medications. Our artificial neural network ANN algorithm exhibited an accuracy of 0.94, specificity of 0.83, sensitivity of 0.97, AUC of 0.96, and MCC of 0.83. These metrics suggest that our model has a high accuracy rate in predicting BBB permeability and therefore has the potential to advance drug discovery efforts in the CNS. This study's outcomes demonstrate the potential for ML models to predict BBB permeability accurately, aiding in the identification of new CNS therapeutic options.Communicated by Ramaswamy H. Sarma.

Keywords:  BBB permeability; Blood–brain barrier; accuracy; area under the curve; artificial neural networks; central nervous system

DOI:  https://doi.org/10.1080/07391102.2024.2326671
J Chem Theory Comput. 2024 Mar 18.

High-Throughput Computational Screening of Solid-Binding Peptides.

Xin Qi, Jim Pfaendtner.

Inspired by biomineralization, a naturally occurring, protein-facilitated process, solid-binding peptides (SBPs) have gained much attention for their potential to fabricate various shaped nanocrystals and hierarchical nanostructures. The advantage of SBPs over other traditionally used synthetic polymers or short ligands is their tunable interaction with the solid material surface via carefully programmed sequence and being solution-dependent simultaneously. However, designing a sequence with targeted binding affinity or selectivity often involves intensive processes such as phage display, and only a limited number of sequences can be identified. Other computational efforts have also been introduced, but the validation process remains prohibitively expensive once a suitable sequence has been identified. In this paper, we present a new model to rapidly estimate the binding free energy of any given sequence to a solid surface. We show how the overall binding of a polypeptide can be estimated from the free energy contribution of each residue based on the statistics of the thermodynamically stable structure ensemble. We validated our model using five silica-binding peptides of different binding affinities and lengths and showed that the model is accurate and robust across a wider range of chemistries and binding strengths. The computational cost of this method can be as low as 3% of the commonly used enhanced sampling scheme for similar studies and has a great potential to be used in high-throughput algorithms to obtain larger training data sets for machine learning SBP screening.

DOI: https://doi.org/10.1021/acs.jctc.3c01286
Nat Commun. 2024 Mar 21. 15(1): 2516

Mutational scanning pinpoints distinct binding sites of key ATGL regulators in lipolysis.

Johanna M Kohlmayr, Gernot F Grabner, Anna Nusser, Anna Höll, Verina Manojlović, Bettina Halwachs, Sarah Masser, Evelyne Jany-Luig, Hanna Engelke, Robert Zimmermann, Ulrich Stelzl.

ATGL is a key enzyme in intracellular lipolysis and plays an important role in metabolic and cardiovascular diseases. ATGL is tightly regulated by a known set of protein-protein interaction partners with activating or inhibiting functions in the control of lipolysis. Here, we use deep mutational protein interaction perturbation scanning and generate comprehensive profiles of single amino acid variants that affect the interactions of ATGL with its regulatory partners: CGI-58, G0S2, PLIN1, PLIN5 and CIDEC. Twenty-three ATGL amino acid variants yield a specific interaction perturbation pattern when validated in co-immunoprecipitation experiments in mammalian cells. We identify and characterize eleven highly selective ATGL switch mutations which affect the interaction of one of the five partners without affecting the others. Switch mutations thus provide distinct interaction determinants for ATGL's key regulatory proteins at an amino acid resolution. When we test triglyceride hydrolase activity in vitro and lipolysis in cells, the activity patterns of the ATGL switch variants trace to their protein interaction profile. In the context of structural data, the integration of variant binding and activity profiles provides insights into the regulation of lipolysis and the impact of mutations in human disease.

DOI: https://doi.org/10.1038/s41467-024-46937-x
bioRxiv. 2024 Mar 08. pii: 2024.03.04.583341. [Epub ahead of print]

Multielectrode array characterization of human induced pluripotent stem cell derived neurons in co-culture with primary human astrocytes.

Maddie R Lemieux, Bernhard Freigassner, Zahra Thathey, Mark R Opp, Charles A Hoeffer, Christopher D Link.

Human induced pluripotent stem cells (hiPSCs) derived into neurons offer a powerful in vitro model to study cellular processes. One method to characterize functional network properties of these cells is using multielectrode arrays (MEAs). MEAs can measure the electrophysiological activity of cellular cultures for extended periods of time without disruption. Here we used WTC11 hiPSCs with a doxycycline-inducible neurogenin 2 (NGN2) transgene differentiated into neurons co-cultured with primary human astrocytes. We achieved a synchrony index ∼0.9 in as little as six-weeks with a mean firing rate of ∼13 Hz. Previous reports show that derived 3D brain organoids can take several months to achieve similar strong network burst synchrony. We also used this co-culture to model aspects of sporadic Alzheimer's disease by mimicking blood-brain barrier breakdown using a human serum. Our fully human co-culture achieved strong network burst synchrony in a fraction of the time of previous reports, making it an excellent first pass, high-throughput method for studying network properties and neurodegenerative diseases.

DOI: https://doi.org/10.1101/2024.03.04.583341
Angew Chem Weinheim Bergstr Ger. 2022 Oct 10. 134(41): e202207508

Enzyme-Activatable Chemokine Conjugates for In Vivo Targeting of Tumor-Associated Macrophages.

Nicole D Barth, Floris J Van Dalen, Utsa Karmakar, Marco Bertolini, Lorena Mendive-Tapia, Takanori Kitamura, Martijn Verdoes, Marc Vendrell.

  Increased levels of tumor-associated macrophages (TAMs) are indicators of poor prognosis in most cancers. Although antibodies and small molecules blocking the recruitment of macrophages to tumors are under evaluation as anticancer therapies, these strategies are not specific for macrophage subpopulations. Herein we report the first enzyme-activatable chemokine conjugates for effective targeting of defined macrophage subsets in live tumors. Our constructs exploit the high expression of chemokine receptors (e.g., CCR2) and the activity of cysteine cathepsins in TAMs to target these cells selectively over other macrophages and immune cells (e.g., neutrophils, T cells, B cells). Furthermore, we demonstrate that cathepsin-activatable chemokines are compatible with both fluorescent and therapeutic cargos, opening new avenues in the design of targeted theranostic probes for immune cells in the tumor microenvironment.

Keywords:  CCL2; Cancer; Cathepsins; Probes; Prodrugs

DOI:  https://doi.org/10.1002/ange.202207508
Adv Exp Med Biol. 2024 ;3234 109-123

The Complementarity of Nuclear Magnetic Resonance and Native Mass Spectrometry in Probing Protein-Protein Interactions.

Elisabetta Boeri Erba, Annalisa Pastore.

  Nuclear magnetic resonance (NMR) and native mass spectrometry (MS) are mature physicochemical techniques with long histories and important applications. NMR spectroscopy provides detailed information about the structure, dynamics, interactions, and chemical environment of biomolecules. MS is an effective approach for determining the mass of biomolecules with high accuracy, sensitivity, and speed. The two techniques offer unique advantages and provide solid tools for structural biology. In the present review, we discuss their individual merits in the context of their applications to structural studies in biology with specific focus on protein interactions and evaluate their limitations. We provide specific examples in which these techniques can complement each other, providing new information on the same scientific case. We discuss how the field may develop and what challenges are expected in the future. Overall, the combination of NMR and MS plays an increasingly important role in integrative structural biology, assisting scientists in deciphering the three-dimensional structure of composite macromolecular assemblies.

Keywords:  Biophysics; Native mass spectrometry (MS); Nuclear magnetic resonance (NMR); Protein interactions; Structural biology

DOI:  https://doi.org/10.1007/978-3-031-52193-5_8
Nat Commun. 2024 Mar 19. 15(1): 2431

The SecM arrest peptide traps a pre-peptide bond formation state of the ribosome.

Felix Gersteuer, Martino Morici, Sara Gabrielli, Keigo Fujiwara, Haaris A Safdari, Helge Paternoga, Lars V Bock, Shinobu Chiba, Daniel N Wilson.

Nascent polypeptide chains can induce translational stalling to regulate gene expression. This is exemplified by the E. coli secretion monitor (SecM) arrest peptide that induces translational stalling to regulate expression of the downstream encoded SecA, an ATPase that co-operates with the SecYEG translocon to facilitate insertion of proteins into or through the cytoplasmic membrane. Here we present the structure of a ribosome stalled during translation of the full-length E. coli SecM arrest peptide at 2.0 Å resolution. The structure reveals that SecM arrests translation by stabilizing the Pro-tRNA in the A-site, but in a manner that prevents peptide bond formation with the SecM-peptidyl-tRNA in the P-site. By employing molecular dynamic simulations, we also provide insight into how a pulling force on the SecM nascent chain can relieve the SecM-mediated translation arrest. Collectively, the mechanisms determined here for SecM arrest and relief are also likely to be applicable for a variety of other arrest peptides that regulate components of the protein localization machinery identified across a wide range of bacteria lineages.

DOI: https://doi.org/10.1038/s41467-024-46762-2
Clin Transl Oncol. 2024 Mar 21.

Current approaches in glioblastoma multiforme immunotherapy.

Marjan Aghajani, Nazila Jalilzadeh, Ali Aghebati-Maleki, Amirhossein Yari, Peyman Tabnak, Amirhossein Mardi, Hossein Saeedi, Leili Aghebati-Maleki, Behzad Baradaran.

  Glioblastoma multiform (GBM) is the most prevalent CNS (central nervous system) tumor in adults, with an average survival length shorter than 2 years and rare metastasis to organs other than CNS. Despite extensive attempts at surgical resecting, the inherently permeable nature of this disease has rendered relapse nearly unavoidable. Thus, immunotherapy is a feasible alternative, as stimulated immune cells can enter into the remote and inaccessible tumor cells. Immunotherapy has revolutionized patient upshots in various malignancies and might introduce different effective ways for GBM patients. Currently, researchers are exploring various immunotherapeutic strategies in patients with GBM to target both the innate and acquired immune responses. These approaches include reprogrammed tumor-associated macrophages, the use of specific antibodies to inhibit tumor progression and metastasis, modifying tumor-associated macrophages with antibodies, vaccines that utilize tumor-specific dendritic cells to activate anti-tumor T cells, immune checkpoint inhibitors, and enhanced T cells that function against tumor cells. Despite these findings, there is still room for improving the response faults of the many currently tested immunotherapies. This study aims to review the currently used immunotherapy approaches with their molecular mechanisms and clinical application in GBM.

Keywords:  Glioblastoma multiforme; Immune checkpoint inhibitor; Immunotherapy

DOI:  https://doi.org/10.1007/s12094-024-03395-7
J Med Chem. 2024 Mar 19.

Identification of Novel Series of Potent and Selective Relaxin Family Peptide Receptor 1 (RXFP1) Agonists.

Kenneth L Granberg, Shigeki Sakamaki, Ryuichi Fuchigami, Yasuki Niwa, Masakazu Fujio, Harutoshi Kato, Fredrik Bergström, Niklas Larsson, Mikael Persson, Inmaculada C Villar, Takuya Fujita, Emiko Sugikawa, Magnus Althage, Naoko Yano, Yoshito Yokoyama, Junpei Kimura, Mark Lal, Hideki Mochida.

Relaxin H2 is a clinically relevant peptide agonist for relaxin family peptide receptor 1 (RXFP1), but a combination of this hormone's short plasma half-life and the need for injectable delivery limits its therapeutic potential. We sought to overcome these limitations through the development of a potent small molecule (SM) RXFP1 agonist. Although two large SM HTS campaigns failed in identifying suitable hit series, we uncovered novel chemical space starting from the only known SM RXFP1 agonist series, represented by ML290. Following a design-make-test-analyze strategy based on improving early dose to man ranking, we discovered compound 42 (AZ7976), a highly selective RXFP1 agonist with sub-nanomolar potency. We used AZ7976, its 10 000-fold less potent enantiomer 43 and recombinant relaxin H2 to evaluate in vivo pharmacology and demonstrate that AZ7976-mediated heart rate increase in rats was a result of RXFP1 agonism. As a result, AZ7976 was selected as lead for continued optimization.

DOI: https://doi.org/10.1021/acs.jmedchem.3c02183
J Med Chem. 2024 Mar 20.

Long-Residence Time Peptide Antagonist for the Vasopressin V2 Receptor to Treat Autosomal Dominant Polycystic Kidney Disease.

Xiaochun Xiong, Naiyuan Wang, Yixiao Zhang, Wenchao Zhao, Ningning Pang, Kequan Fu, Nan Zhou, Xueyan Zhou, Dong Guo.

The dysregulated intracellular cAMP in the kidneys drives cystogenesis and progression in autosomal dominant polycystic kidney disease (ADPKD). Mounting evidence supports that vasopressin V2 receptor (V2R) antagonism effectively reduces cAMP levels, validating this receptor as a therapeutic target. Tolvaptan, an FDA-approved V2R antagonist, shows limitations in its clinical efficacy for ADPKD treatment. Therefore, the pursuit of better-in-class V2R antagonists with an improved efficacy remains pressing. Herein, we synthesized a set of peptide V2R antagonists. Peptide 33 exhibited a high binding affinity for the V2R (Ki = 6.1 ± 1.5 nM) and an extended residence time of 20 ± 1 min, 2-fold that of tolvaptan. This prolonged interaction translated into sustained suppression of cAMP production in washout experiments. Furthermore, peptide 33 exhibited improved efficacies over tolvaptan in both ex vivo and in vivo models of ADPKD, underscoring its potential as a promising lead compound for the treatment of ADPKD.

DOI: https://doi.org/10.1021/acs.jmedchem.4c00217
Bull Math Biol. 2024 Mar 19. 86(4): 43

Impact of Resistance on Therapeutic Design: A Moran Model of Cancer Growth.

Mason S Lacy, Adrianne L Jenner.

  Resistance of cancers to treatments, such as chemotherapy, largely arise due to cell mutations. These mutations allow cells to resist apoptosis and inevitably lead to recurrence and often progression to more aggressive cancer forms. Sustained-low dose therapies are being considered as an alternative over maximum tolerated dose treatments, whereby a smaller drug dosage is given over a longer period of time. However, understanding the impact that the presence of treatment-resistant clones may have on these new treatment modalities is crucial to validating them as a therapeutic avenue. In this study, a Moran process is used to capture stochastic mutations arising in cancer cells, inferring treatment resistance. The model is used to predict the probability of cancer recurrence given varying treatment modalities. The simulations predict that sustained-low dose therapies would be virtually ineffective for a cancer with a non-negligible probability of developing a sub-clone with resistance tendencies. Furthermore, calibrating the model to in vivo measurements for breast cancer treatment with Herceptin, the model suggests that standard treatment regimens are ineffective in this mouse model. Using a simple Moran model, it is possible to explore the likelihood of treatment success given a non-negligible probability of treatment resistant mutations and suggest more robust therapeutic schedules.

Keywords:  Cancer; Moran model; Resistance; Treatment

DOI:  https://doi.org/10.1007/s11538-024-01272-6