bims-cepepe 2023-12-24 papers

bims-cepepe

Biomed News

on Cell-penetrating peptides

Issue of 2023–12–24
seventeen papers selected by
Henry Lamb, Queensland University of Technology

Cell-Penetrating Peptide-Bismuth Bicycles.
Stabilized cyclic peptides as modulators of protein-protein interactions: promising strategies and biological evaluation.
Peptide toxins that target vertebrate voltage-gated sodium channels underly the painful stings of harvester ants.
Unlocking novel therapies: cyclic peptide design for amyloidogenic targets through synergies of experiments, simulations, and machine learning.
A Method for Using Cell-Penetrating Peptides for Loading Plasmid DNA into Secreted Extracellular Vesicles.
Development, Characterization, and Radiation Dosimetry Studies of 18F-BMS-986229, a 18F-Labeled PD-L1 Macrocyclic Peptide PET Tracer.
De novo design of high-affinity binders of bioactive helical peptides.
Recent advancements on in vitro blood-brain barrier model: a reliable and efficient screening approach for preclinical and clinical investigation.
Exploring Cyclic Peptide Nanotube Stability Across Diverse Lipid Bilayers and Unveiling Water Transport Dynamics.
Putting peptides on display.
The ERK inhibitor LY3214996 augments anti-PD-1 immunotherapy in preclinical mouse models of BRAFV600E melanoma brain metastasis.
Development and validation of PAMPA-BBB QSAR model to predict brain penetration potential of novel drug candidates.
Molecular chameleons in drug discovery.
Exploring the synthetic approaches and clinical prowess of established macrocyclic pharmaceuticals.
Quantification of Binding of Small Molecules to Native Proteins Overexpressed in Living Cells.
The Marine Natural Compound Dragmacidin D Selectively Induces Apoptosis in Triple-Negative Breast Cancer Spheroids.
Characterization of an iPSC-based barrier model for blood-brain barrier investigations using the SBAD0201 stem cell line.

Angew Chem Int Ed Engl. 2023 Dec 21. e202318615

Cell-Penetrating Peptide-Bismuth Bicycles.

Saan Voss, Liam Adair, Katharina Achazi, Heeyoung Kim, Silke Bergemann, Ralf Bartenschlager, Elizabeth New, Jörg Rademann, Christoph Nitsche.

  Cell-penetrating peptides (CPPs) play a significant role in the delivery of cargos into human cells. We report the first CPPs based on peptide-bismuth bicycles, which can be readily obtained from commercially available peptide precursors, making them accessible for a wide range of applications. These CPPs enter mammalian cells as demonstrated by live-cell microscopy using fluorescently labelled peptides. We report efficient sequences that demonstrate increased cellular uptake compared to conventional CPPs like the TAT peptide (derived from the transactivating transcriptional activator of human immunodeficiency virus 1) or octaarginine (R8), despite requiring only three positive charges. Bicyclization triggered by the presence of bismuth(III) increases cellular uptake by more than one order of magnitude. Through the analysis of cell lysates using inductive coupled plasma mass spectrometry (ICP-MS), we have introduced an alternative approach to examine the cellular uptake of CPPs. This has allowed us to confirm the presence of bismuth in cells after exposure to our CPPs. Mechanistic studies indicated an energy-dependent endocytic cellular uptake sensitive to inhibition by rottlerin, most likely involving macropinocytosis.

Keywords:  Fluorescence; Peptides; bicycles; bismuth; cells

DOI:  https://doi.org/10.1002/anie.202318615
RSC Med Chem. 2023 Dec 13. 14(12): 2496-2508

Stabilized cyclic peptides as modulators of protein-protein interactions: promising strategies and biological evaluation.

Jiongjia Cheng, Junlong Zhou, Lingyan Kong, Haiying Wang, Yuchi Zhang, Xiaofeng Wang, Guangxiang Liu, Qian Chu.

Protein-protein interactions (PPIs) control many essential biological pathways which are often misregulated in disease. As such, selective PPI modulators are desirable to unravel complex functions of PPIs and thus expand the repertoire of therapeutic targets. However, the large size and relative flatness of PPI interfaces make them challenging molecular targets for conventional drug modalities, rendering most PPIs "undruggable". Therefore, there is a growing need to discover innovative molecules that are able to modulate crucial PPIs. Peptides are ideal candidates to deliver such therapeutics attributed to their ability to closely mimic structural features of protein interfaces. However, their inherently poor proteolysis resistance and cell permeability inevitably hamper their biomedical applications. The introduction of a constraint (i.e., peptide cyclization) to stabilize peptides' secondary structure is a promising strategy to address this problem as witnessed by the rapid development of cyclic peptide drugs in the past two decades. Here, we comprehensively review the recent progress on stabilized cyclic peptides in targeting challenging PPIs. Technological advancements and emerging chemical approaches for stabilizing active peptide conformations are categorized in terms of α-helix stapling, β-hairpin mimetics and macrocyclization. To discover potent and selective ligands, cyclic peptide library technologies were updated based on genetic, biochemical or synthetic methodologies. Moreover, several advances to improve the permeability and oral bioavailability of biologically active cyclic peptides enable the de novo development of cyclic peptide ligands with pharmacological properties. In summary, the development of cyclic peptide-based PPI modulators carries tremendous promise for the next generation of therapeutic agents to target historically "intractable" PPI systems.

DOI: https://doi.org/10.1039/d3md00487b
J Biol Chem. 2023 Dec 16. pii: S0021-9258(23)02605-4. [Epub ahead of print] 105577

Peptide toxins that target vertebrate voltage-gated sodium channels underly the painful stings of harvester ants.

Samuel D Robinson, Jennifer R Deuis, Pancong Niu, Axel Touchard, Alexander Mueller, Vanessa Schendel, Nina Brinkwirth, Glenn F King, Irina Vetter, Justin O Schmidt.

  Harvester ants (genus Pogonomyrmex) are renowned for their stings which cause intense, long-lasting pain and other neurotoxic symptoms in vertebrates. Here we show that harvester ant venoms are relatively simple and composed largely of peptide toxins. One class of peptides is primarily responsible for the long-lasting local pain of envenomation via activation of peripheral sensory neurons. These hydrophobic, cysteine-free peptides potently modulate mammalian voltage-gated sodium (NaV) channels, reducing the voltage threshold for activation and inhibiting channel inactivation. These toxins appear to have evolved specifically to deter vertebrates.

Keywords:  Ion channel; Pain; Pogonomyrmex; Venom

DOI:  https://doi.org/10.1016/j.jbc.2023.105577
Chem Commun (Camb). 2023 Dec 22.

Unlocking novel therapies: cyclic peptide design for amyloidogenic targets through synergies of experiments, simulations, and machine learning.

Daria de Raffele, Ioana M Ilie.

Existing therapies for neurodegenerative diseases like Parkinson's and Alzheimer's address only their symptoms and do not prevent disease onset. Common therapeutic agents, such as small molecules and antibodies struggle with insufficient selectivity, stability and bioavailability, leading to poor performance in clinical trials. Peptide-based therapeutics are emerging as promising candidates, with successful applications for cardiovascular diseases and cancers due to their high bioavailability, good efficacy and specificity. In particular, cyclic peptides have a long in vivo stability, while maintaining a robust antibody-like binding affinity. However, the de novo design of cyclic peptides is challenging due to the lack of long-lived druggable pockets of the target polypeptide, absence of exhaustive conformational distributions of the target and/or the binder, unknown binding site, methodological limitations, associated constraints (failed trials, time, money) and the vast combinatorial sequence space. Hence, efficient alignment and cooperation between disciplines, and synergies between experiments and simulations complemented by popular techniques like machine-learning can significantly speed up the therapeutic cyclic-peptide development for neurodegenerative diseases. We review the latest advancements in cyclic peptide design against amyloidogenic targets from a computational perspective in light of recent advancements and potential of machine learning to optimize the design process. We discuss the difficulties encountered when designing novel peptide-based inhibitors and we propose new strategies incorporating experiments, simulations and machine learning to design cyclic peptides to inhibit the toxic propagation of amyloidogenic polypeptides. Importantly, these strategies extend beyond the mere design of cyclic peptides and serve as template for the de novo generation of (bio)materials with programmable properties.

DOI: https://doi.org/10.1039/d3cc04630c
Biomolecules. 2023 Dec 06. pii: 1751. [Epub ahead of print]13(12):

A Method for Using Cell-Penetrating Peptides for Loading Plasmid DNA into Secreted Extracellular Vesicles.

Jekaterina Nebogatova, Heleri Heike Härk, Anett Puskar, Ly Porosk, Paolo Guazzi, Moataz Dowaidar, Ülo Langel, Kaido Kurrikoff.

  The low bioavailability and high toxicity of plasmid DNA (pDNA)-based therapeutics pose challenges for their in vivo application. Extracellular vesicles (EVs) have great potential to overcome these limitations, as they are biocompatible native cargo carriers. Various methods for loading pDNA into EVs, including electroporation, sonication, and co-incubation, have been previously investigated, but their success has been questionable. In this study, we report a unique method for loading EVs with pDNA through transient transfection using cell-penetrating peptides (CPPs). With this method, we found a 104-fold increase in the expression levels of the luciferase reporter protein in recipient cells compared to the untreated cells. These data point to the high transfection efficacy and bioavailability of the delivered encapsulated nucleic acid. Furthermore, the in vivo experimental data indicate that the use of pDNA-loaded EVs as native delivery vehicles reduces the toxic effects associated with traditional nucleic acid (NA) delivery and treatment.

Keywords:  cell-penetrating peptides; drug delivery system; extracellular vesicles; gene therapy; nucleic acid therapeutics

DOI:  https://doi.org/10.3390/biom13121751
Mol Imaging Biol. 2023 Dec 20.

Development, Characterization, and Radiation Dosimetry Studies of 18F-BMS-986229, a 18F-Labeled PD-L1 Macrocyclic Peptide PET Tracer.

Joonyoung Kim, David J Donnelly, Tritin Tran, Adrienne Pena, Andrea Olga Shorts, Thomas V Petrone, Yunhui Zhang, Kenneth M Boy, Paul M Scola, Daniel J Tenney, Michael A Poss, Matthew G Soars, Samuel J Bonacorsi, Erin L Cole, Diederik J Grootendorst, Patrick L Chow, Nicholas A Meanwell, Shuyan Du.

   PURPOSE: In cancer immunotherapy, the blockade of the interaction between programmed death-1 and its ligand (PD-1:PD-L1) has proven to be one of the most promising strategies. However, as mechanisms of resistance to PD-1/PD-L1 inhibition include variability in tumor cell PD-L1 expression in addition to standard tumor biopsy PD-L1 immunohistochemistry (IHC), a comprehensive and quantitative approach for measuring PD-L1 expression is required. Herein, we report the development and characterization of an 18F-PD-L1-binding macrocyclic peptide as a PET tracer for the comprehensive evaluation of tumor PD-L1 expression in cancer patients.
PROCEDURES: 18F-BMS-986229 was characterized for PD-L1 expression assessment by autoradiography or PET imaging. 18F-BMS-986229 was utilized to evaluate tumor PD-L1 target engagement in competition with a macrocyclic peptide inhibitor of PD-L1 (BMS-986189) over a range of doses using PET imaging. A whole-body radiation dosimetry study of 18F-BMS-986229 in healthy non-human primates (NHPs) was performed.
RESULTS: In vitro autoradiography showed an 8:1 binding ratio in L2987(PD-L1 +) vs. HT-29 (PD-L1-) tumors, more than 90% of which could be blocked with 1 nM of BMS-986189. Ex vivo autoradiography showed that 18F-BMS-986229 detection was penetrant over a series of sections spanning the entire L2987 tumor. In vivo PET imaging in mice demonstrated a 5:1 tracer uptake ratio (at 90-100 min after tracer administration) in L2987 vs. HT-29 tumors and demonstrated 83%-93% specific binding of BMS-986189 within those dose ranges. In a healthy NHP dosimetry study, the resultant whole-body effective dose was 0.025 mSv/MBq.
CONCLUSION: 18F-BMS-986229 has been preclinically characterized and exhibits high target specificity, low background uptake, and a short blood half-life supportive of same day imaging in the clinic. As the PET tracer, 18F-BMS-986229 shows promise in the quantification of PD-L1 expression, and its use in monitoring longitudinal changes in patients may provide insights into PD-1:PD-L1 immuno-therapy treatment outcomes.

Keywords:  18F-labeled macrocyclic peptide; PD-1:PD-L1 checkpoint inhibitor; Preclinical PET imaging

DOI:  https://doi.org/10.1007/s11307-023-01889-4
Nature. 2023 Dec 18.

De novo design of high-affinity binders of bioactive helical peptides.

Susana Vázquez Torres, Philip J Y Leung, Preetham Venkatesh, Isaac D Lutz, Fabian Hink, Huu-Hien Huynh, Jessica Becker, Andy Hsien-Wei Yeh, David Juergens, Nathaniel R Bennett, Andrew N Hoofnagle, Eric Huang, Michael J MacCoss, Marc Expòsit, Gyu Rie Lee, Asim K Bera, Alex Kang, Joshmyn De La Cruz, Paul M Levine, Xinting Li, Mila Lamb, Stacey R Gerben, Analisa Murray, Piper Heine, Elif Nihal Korkmaz, Jeff Nivala, Lance Stewart, Joseph L Watson, Joseph M Rogers, David Baker.

Many peptide hormones form an alpha-helix upon binding their receptors1-4, and sensitive detection methods for them could contribute to better clinical management of disease5. De novo protein design can now generate binders with high affinity and specificity to structured proteins6,7. However, the design of interactions between proteins and short peptides with helical propensity is an unmet challenge. Here, we describe parametric generation and deep learning-based methods for designing proteins to address this challenge. We show that by extending RFdiffusion8 to enable binder design to flexible targets, and to refining input structure models by successive noising and denoising (partial diffusion), picomolar affinity binders can be generated to helical peptide targets both by refining designs generated with other methods, or completely de novo starting from random noise distributions. To our knowledge these are the highest affinity designed binding proteins against any protein or small molecule target generated directly by computation without any experimental optimisation. The RFdiffusion designs enable the enrichment and subsequent detection of parathyroid hormone and glucagon by mass spectrometry, and the construction of bioluminescence-based protein biosensors. The ability to design binders to conformationally variable targets, and to optimise by partial diffusion both natural and designed proteins, should be broadly useful.

DOI: https://doi.org/10.1038/s41586-023-06953-1
Expert Opin Drug Deliv. 2023 Dec 15.

Recent advancements on in vitro blood-brain barrier model: a reliable and efficient screening approach for preclinical and clinical investigation.

Snigdha Singh, Mukta Agrawal, Rajat Vashist, Rohit K Patel, Sanchita Dhanchandra Sangave, Amit Alexander.

   INTRODUCTION: The efficiency of brain therapeutics is greatly hindered by the blood-brain barrier (BBB). BBB's protective function, selective permeability, and dynamic functionality maintain the harmony between the brain and peripheral region. Thus, the design of any novel drug carrier system requires the complete study and investigation of BBB permeability, efflux transport, and the effect of associated cellular and non-vascular unit trafficking on BBB penetrability. The in vitro BBB models offer a most promising, and reliable mode of initial investigation of BBB permeability and associated factors as strong evidence for further preclinical and clinical investigation.
AREA COVERED: This review work covers the structure and functions of BBB components and different types of in vitro BBB models along with factors affecting BBB model development and model selection criteria.
EXPERT OPINION: In vivo models assume to reciprocate the physiological environment to the maximum extent. However, the interspecies variability, NVUs trafficking, dynamic behavior of BBB, etc. lead to non-reproducible results. The in vitro models are comparatively less complex, and flexible, as per the study design, could generate substantial evidence and help identify suitable in vivo animal model selection.

Keywords:  BBB-on-chip; In vitro BBB model; Microfluidic design; Stem cell-derived BBB model; model selection criterion

DOI:  https://doi.org/10.1080/17425247.2023.2295940
Langmuir. 2023 Dec 22.

Exploring Cyclic Peptide Nanotube Stability Across Diverse Lipid Bilayers and Unveiling Water Transport Dynamics.

Rimjhim Moral, Sandip Paul.

Cyclic Peptide Nanotubes (CPNTs) have emerged as compelling candidates for various applications, particularly as nanochannels within lipid bilayers. In this study, the stability of two CPNTs, namely 8 × [(Cys-Gly-Met-Gly)2] and 8 × [(Gly-Leu)4], are comprehensively investigated across different lipid bilayers, including 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), a mixed model membrane (POPE/POPG), and a realistic yeast model membrane. The results demonstrate that both CPNTs maintain their tubular structures in all lipid bilayers, with [(Cys-Gly-Met-Gly)2] showing increased stability over an extended period in these lipid membranes. The insertion of CPNTs shows negligible impact on lipid bilayer properties, including area per lipid, volume per lipid, and bilayer thickness. The study demonstrates that the CPNT preserves its two-line water movement pattern within all the lipid membranes, reaffirming their potential as water channels. The MSD curves further reveal that the dynamics of water molecules inside the nanotube are similar for all the bilayer systems with minor differences that arise due to different lipid environments.

DOI: https://doi.org/10.1021/acs.langmuir.3c03030
Nat Chem Biol. 2024 Jan;20(1): 1-2

Putting peptides on display.

Russell Johnson.

DOI: https://doi.org/10.1038/s41589-023-01494-0
Neuro Oncol. 2023 Dec 22. pii: noad248. [Epub ahead of print]

The ERK inhibitor LY3214996 augments anti-PD-1 immunotherapy in preclinical mouse models of BRAFV600E melanoma brain metastasis.

Magali A de Sauvage, Consuelo Torrini, Edwin Nieblas-Bedolla, Elizabeth Summers, Emily Sullivan, Britney S Zhang, Emily Batchelor, Braxton Marion, Erika Yamazawa, Samuel C Markson, Hiroaki Wakimoto, Naema Nayyar, Priscilla Brastianos.

   BACKGROUND: Immune checkpoint inhibitors (ICI) have revolutionized cancer treatment; however, only a subset of patients with brain metastasis (BM) respond to ICI. Activating mutations in the mitogen-activated protein kinase (MAPK) signaling pathway are frequent in BM. The objective of this study was to evaluate whether therapeutic ERK inhibition can improve the efficacy of ICI for BM.
METHODS: We used immunotypical mouse models of brain metastasis bearing dual extracranial/intracranial tumors to evaluate efficacy of single-agent and dual-agent treatment with selective ERK inhibitor LY3214996 (LY321) and anti-PD-1 antibody. We verified target inhibition and drug delivery and investigated treatment-induced changes in T cell response and tumor-immune microenvironment using high-parameter flow cytometry, multiplex immunoassays and T cell receptor profiling.
RESULTS: We found that dual treatment with LY321 and anti-PD-1 significantly improved overall survival in two BRAFV600E-mutant murine melanoma models but not in KRAS-mutant murine lung adenocarcinoma. We demonstrate that although LY321 has limited blood brain barrier (BBB) permeability, combined LY321 and anti-PD-1 therapy increases tumor-infiltrating CD8+ effector T cells, broadens the T cell receptor repertoire in the extracranial tumor, enriches T cell clones shared by the periphery and brain, and reduces immunosuppressive cytokines and cell populations in tumors.
CONCLUSIONS: Despite limited BBB permeability of LY321, combination LY321 and anti-PD-1 treatment amplifies extracranial immune responses that improve intracranial disease control, highlighting the role of extracranial tumors in driving intracranial response to treatment. Combined ERK and PD-1 inhibition is a promising therapeutic approach, worthy of further investigation for patients with melanoma BM.

Keywords:  Brain metastasis; Combination treatment; Immune checkpoint inhibitors; MAPK; Preclinical

DOI:  https://doi.org/10.1093/neuonc/noad248
Front Pharmacol. 2023 ;14 1291246

Development and validation of PAMPA-BBB QSAR model to predict brain penetration potential of novel drug candidates.

Rintaro Kato, Wenyu Zeng, Vishal B Siramshetty, Jordan Williams, Md Kabir, Natalie Hagen, Elias C Padilha, Amy Q Wang, Ewy A Mathé, Xin Xu, Pranav Shah.

  Efficiently circumventing the blood-brain barrier (BBB) poses a major hurdle in the development of drugs that target the central nervous system. Although there are several methods to determine BBB permeability of small molecules, the Parallel Artificial Membrane Permeability Assay (PAMPA) is one of the most common assays in drug discovery due to its robust and high-throughput nature. Drug discovery is a long and costly venture, thus, any advances to streamline this process are beneficial. In this study, ∼2,000 compounds from over 60 NCATS projects were screened in the PAMPA-BBB assay to develop a quantitative structure-activity relationship model to predict BBB permeability of small molecules. After analyzing both state-of-the-art and latest machine learning methods, we found that random forest based on RDKit descriptors as additional features provided the best training balanced accuracy (0.70 ± 0.015) and a message-passing variant of graph convolutional neural network that uses RDKit descriptors provided the highest balanced accuracy (0.72) on a prospective validation set. Finally, we correlated in vitro PAMPA-BBB data with in vivo brain permeation data in rodents to observe a categorical correlation of 77%, suggesting that models developed using data from PAMPA-BBB can forecast in vivo brain permeability. Given that majority of prior research has relied on in vitro or in vivo data for assessing BBB permeability, our model, developed using the largest PAMPA-BBB dataset to date, offers an orthogonal means to estimate BBB permeability of small molecules. We deposited a subset of our data into PubChem bioassay database (AID: 1845228) and deployed the best performing model on the NCATS Open Data ADME portal (https://opendata.ncats.nih.gov/adme/). These initiatives were undertaken with the aim of providing valuable resources for the drug discovery community.

Keywords:  ADME; blood-brain barrier (BBB); computational drug design; drug discovery; parallel artificial membrane permeability assay (PAMPA); quantitative structure-activity relationship (QSAR)

DOI:  https://doi.org/10.3389/fphar.2023.1291246
Nat Rev Chem. 2023 Dec 20.

Molecular chameleons in drug discovery.

Vasanthanathan Poongavanam, Lianne H E Wieske, Stefan Peintner, Máté Erdélyi, Jan Kihlberg.

Molecular chameleons possess a flexibility that allows them to dynamically shield or expose polar functionalities in response to the properties of the environment. Although the concept of molecular chameleons was introduced already in 1970, interest in them has grown considerably since the 2010s, when drug discovery has focused to an increased extent on new chemical modalities. Such modalities include cyclic peptides, macrocycles and proteolysis-targeting chimeras, all of which reside in a chemical space far from that of traditional small-molecule drugs. Both cell permeability and aqueous solubility are required for the oral absorption of drugs. Engineering these properties, and potent target binding, into the larger new modalities is a more daunting task than for traditional small-molecule drugs. The ability of chameleons to adapt to different environments may be essential for success. In this Review, we provide both general and theoretical insights into the realm of molecular chameleons. We discuss why chameleons have come into fashion and provide a do-it-yourself toolbox for their design; we then provide a glimpse of how advanced in silico methods can support molecular chameleon design.

DOI: https://doi.org/10.1038/s41570-023-00563-1
Eur J Med Chem. 2023 Dec 14. pii: S0223-5234(23)01018-8. [Epub ahead of print]264 116051

Exploring the synthetic approaches and clinical prowess of established macrocyclic pharmaceuticals.

Jian-Gang Sun, Peng Nie, Piet Herdewijn, Xiao-Jing Li.

  Macrocyclic compounds, characterized by cyclic structures, often originate from either modified forms of unicyclic canonical molecules or natural products. Within the field of medicinal chemistry, there has been a growing fascination with drug-like macrocycles in recent years, primarily due to compelling evidence indicating that macrocyclization can significantly influence both the biological and physiochemical properties, as well as the selectivity, when compared to their acyclic counterparts. The approval of contemporary pharmaceutical agents like Lorlatinib underscore the notable clinical relevance of drug-like macrocycles. Nonetheless, the synthesis of these drug-like macrocycles poses substantial challenges, primarily stemming from the complexity of ring-closing reactions, which are inherently dependent on the size and geometry of the bridging linker, impacting overall yields. Nevertheless, macrocycles offer a promising avenue for expanding the synthetic toolkit in medicinal chemistry, enabling the creation of bioactive compounds. To shed light on the subject, we delve into the clinical prowess of established macrocyclic drugs, spanning various therapeutic areas, including oncology, and infectious diseases. Case studies of clinically approved macrocyclic agents illustrate their profound impact on patient care and disease management. As we embark on this journey through the world of macrocyclic pharmaceuticals, we aim to provide a comprehensive overview of their synthesis and clinical applications, shedding light on the pivotal role they play in modern medicine.

Keywords:  Clinical applications; Drugs; Macrocyclic pharmaceuticals; Synthetic approaches

DOI:  https://doi.org/10.1016/j.ejmech.2023.116051
J Am Chem Soc. 2023 Dec 20.

Quantification of Binding of Small Molecules to Native Proteins Overexpressed in Living Cells.

Yuwen Yin, Serena Li Zhao, Digamber Rane, Zhihong Lin, Meng Wu, Blake R Peterson.

The affinity and selectivity of small molecules for proteins drive drug discovery and development. We report a fluorescent probe cellular binding assay (FPCBA) for determination of these values for native (untagged) proteins overexpressed in living cells. This method uses fluorophores such as Pacific Blue (PB) linked to cell-permeable protein ligands to generate probes that rapidly and reversibly equilibrate with intracellular targets, as established by kinetic assays of cellular uptake and efflux. To analyze binding to untagged proteins, an internal ribosomal entry site (IRES) vector was employed that allows a single mRNA to encode both the protein target and a separate orthogonal fluorescent protein (mVenus). This enabled cellular uptake of the probe to be correlated with protein expression by flow cytometry, allowing measurement of cellular dissociation constants (Kd) of the probe. This approach was validated by studies of the binding of allosteric activators to eight different Protein Kinase C (PKC) isozymes. Full-length PKCs expressed in transiently transfected HEK293T cells were used to measure cellular Kd values of a probe comprising PB linked to the natural product phorbol via a carbamate. These values were further used to determine competitive binding constants (cellular Ki values) of the nonfluorescent phorbol ester PDBu and the anticancer agent bryostatin 1 for each isozyme. For some PKC-small molecule pairs, these cellular Ki values matched known biochemical Ki values, but for others, altered selectivity was observed in cells. This approach can facilitate quantification of interactions of small molecules with physiologically relevant native proteins.

DOI: https://doi.org/10.1021/jacs.3c07488
Mar Drugs. 2023 Dec 15. pii: 642. [Epub ahead of print]21(12):

The Marine Natural Compound Dragmacidin D Selectively Induces Apoptosis in Triple-Negative Breast Cancer Spheroids.

Esther A Guzmán, Tara A Peterson, Amy E Wright.

  Cancer cells grown in 3D spheroid cultures are considered more predictive for clinical efficacy. The marine natural product dragmacidin D induces apoptosis in MDA-MB-231 and MDA-MB-468 triple-negative breast cancer (TNBC) spheroids within 24 h of treatment while showing no cytotoxicity against the same cells grown in monolayers and treated for 72 h. The IC50 for cytotoxicity based on caspase 3/7 cleavage in the spheroid assay was 8 ± 1 µM in MDA-MB-231 cells and 16 ± 0.6 µM in MDA-MB-468 cells at 24 h. No cytotoxicity was seen at all in 2D, even at the highest concentration tested. Thus, the IC50 for cytotoxicity in the MTT assay (2D) in these cells was found to be >75 µM at 72 h. Dragmacidin D exhibited synergy when used in conjunction with paclitaxel, a current treatment for TNBC. Studies into the signaling changes using a reverse-phase protein array showed that treatment with dragmacidin D caused significant decreases in histones. Differential protein expression was used to hypothesize that its potential mechanism of action involves acting as a protein synthesis inhibitor or a ribonucleotide reductase inhibitor. Further testing is necessary to validate this hypothesis. Dragmacidin D also caused a slight decrease in an invasion assay in the MDA-MB-231 cells, although this failed to be statistically significant. Dragmacidin D shows intriguing selectivity for spheroids and has the potential to be a treatment option for triple-negative breast cancer, which merits further research into understanding this activity.

Keywords:  apoptosis; invasion; marine natural products; reverse-phase protein arrays; spheroids; triple-negative breast cancer

DOI:  https://doi.org/10.3390/md21120642
Fluids Barriers CNS. 2023 Dec 19. 20(1): 96

Characterization of an iPSC-based barrier model for blood-brain barrier investigations using the SBAD0201 stem cell line.

Burak Ozgür, Elena Puris, Andreas Brachner, Antje Appelt-Menzel, Sabrina Oerter, Viktor Balzer, Mikkel Roland Holst, Rasmus Folmann Christiansen, Kathrine Hyldig, Stephen T Buckley, Mie Kristensen, Seppo Auriola, Allan Jensen, Gert Fricker, Morten Schallburg Nielsen, Winfried Neuhaus, Birger Brodin.

   BACKGROUND: Blood-brain barrier (BBB) models based on primary murine, bovine, and porcine brain capillary endothelial cell cultures have long been regarded as robust models with appropriate properties to examine the functional transport of small molecules. However, species differences sometimes complicate translating results from these models to human settings. During the last decade, brain capillary endothelial-like cells (BCECs) have been generated from stem cell sources to model the human BBB in vitro. The aim of the present study was to establish and characterize a human BBB model using human induced pluripotent stem cell (hiPSC)-derived BCECs from the hIPSC line SBAD0201.
METHODS: The model was evaluated using transcriptomics, proteomics, immunocytochemistry, transendothelial electrical resistance (TEER) measurements, and, finally, transport assays to assess the functionality of selected transporters and receptor (GLUT-1, LAT-1, P-gp and LRP-1).
RESULTS: The resulting BBB model displayed an average TEER of 5474 ± 167 Ω·cm2 and cell monolayer formation with claudin-5, ZO-1, and occludin expression in the tight junction zones. The cell monolayers expressed the typical BBB markers VE-cadherin, VWF, and PECAM-1. Transcriptomics and quantitative targeted absolute proteomics analyses revealed that solute carrier (SLC) transporters were found in high abundance, while the expression of efflux transporters was relatively low. Transport assays using GLUT-1, LAT-1, and LRP-1 substrates and inhibitors confirmed the functional activities of these transporters and receptors in the model. A transport assay suggested that P-gp was not functionally expressed in the model, albeit antibody staining revealed that P-gp was localized at the luminal membrane.
CONCLUSIONS: In conclusion, the novel SBAD0201-derived BBB model formed tight monolayers and was proven useful for studies investigating GLUT-1, LAT-1, and LRP-1 mediated transport across the BBB. However, the model did not express functional P-gp and thus is not suitable for the performance of drug efflux P-gp reletated studies.

Keywords:  Blood-brain barrier (BBB); Brain capillary endothelial-like cells (BCECs); Efflux transporters; Human induced pluripotent stem cells (hiPSCs); Solute carriers (SLC) transporters; Tight junctions

DOI:  https://doi.org/10.1186/s12987-023-00501-9