Intracellular Targets Research Articles

Photoprotection: Current developments and controversies.

This review aimed at summarizing some of the key points that were discussed during the photoprotection session at the International Forum of Dermatology in 2022. This international conference was designed to address prominent topics of clinical dermatology in a holistic way, allowing to articulate multiple viewpoints. Therefore, this review does not claim to be exhaustive, but is instead intended to give an overview of recent developments and ongoing controversies in the field of photoprotection. Cumulative ultraviolet radiation (UVR) exposure is the major aetiological factor in the development of photoageing, photoimunosuppression and photocarcinogenesis. UVA (320-400 nm) penetrates into the dermis and damages DNA and other intracellular and acellular targets primarily by generating reactive oxygen species (ROS). It is the major contributor to photoageing, characterized by fine and coarse wrinkles, dyspigmentation and loss of elasticity. UVB (290-320 nm) is responsible for sunburns through direct damage to DNA by the formation of 6-4 cyclobutane pyrimidine dimers (CPDs) and pyrimidine 6-4 pyrimidone photoproducts. Both UVA and UVB exposure increase the risk of basal cell carcinoma, squamous cell carcinoma and melanoma. In recent years, visible light (VL; 400-700 nm) has also been implicated in the exacerbation of conditions aggravated by sun exposure such as hyperpigmentation and melasma. Photoprotection is a critical health strategy to reduce the deleterious effects of UVR and VL. Comprehensive photoprotection strategies include staying in the shade when outdoors, wearing photoprotective clothing including a wide-brimmed hat, and sunglasses, and the use of sunscreen. Due to the absorption of UV filters, the safety of sunscreens has been questioned. Newer sunscreens are becoming available with filters with absorption even beyond the UV spectrum, offering enhanced protection compared with older products. Prevention of photocarcinogenesis, sun-induced or sunlight-exacerbated hyperpigmentary conditions and drug-induced photosensitivity is an important reason for adopting comprehensive photoprotection strategies.

Shape Engineering of Exosomes for Endoplasmic Reticulum-Targeted Delivery and Amplified Anticancer Efficacy

Exosomes are nanoscale extracellular vesicles that have become pivotal in advancing targeted drug delivery strategies for cancer therapy. In this study, we conducted a comparative analysis of the intracellular targeting capabilities of differently shaped exosomes, including milk exosome nanorods (MR), ginger exosome nanorods (GR), and cancer cell exosome nanorods (HR), compared to their spherical counterparts. Our observations revealed that exosome nanorods demonstrated effective and sustained targeting of the endoplasmic reticulum (ER) within cancer cells, while exosome nanospheres were captured within lysosomes. Building on this principle, we chose milk-derived exosomes (mExo) for the in vivo research, engineering the surface of MR with folate to enhance their tumor-targeting efficacy. We demonstrated the effective accumulation of these folate-modified MR (FMR) around the ER in cancer cells, as validated in both orthotopic colorectal cancer (CRC) tissues and human CRC biopsy samples. Furthermore, when loaded with curcumin (Cur), the FMR@Cur exhibited remarkable efficacy in suppressing tumors in orthotopic CRC mouse models. This effect is attributed to the targeted delivery of FMR@Cur to the ER, leading to enhanced ER-stress induced apoptosis. Overall, our study underscores the pivotal role of shape engineering in exosome-mediated drug delivery, offering novel insights and paving the way for innovative strategies to enhance the precision of intracellular drug targeting in cancer therapy.

Triple-transformable dynamic surroundings for programmed transportation of bio-vulnerable mRNA payloads towards systemic treatment of intractable solid tumors

The surface physiochemical properties of nanomedicine play a crucial role in modulating biointerfacial reactions in sequential biological compartments, accordingly accomplishing the desired programmed delivery scenario to intracellular targets. PEGylation, which involves modifying the surface with a layer of poly(ethylene glycol), has been validated as an effective strategy for minimizing adverse biointerfacial interactions. However, it has also been observed to impede cellular uptake and intracellular trafficking activities. To address this dilemma, we propose a dynamic surface chemistry approach that actively prevents non-specific reactions in systemic circulation, while readily facilitating cellular uptake by converting into a highly cytomembrane-adhesive state. Moreover, the surface becomes more adhesive to endolysosomal membranes, enabling translocation into the cytosol. In this study, PEGylated mRNA delivery nanoparticulates were tethered with charge-reversible polymers to create dynamic surroundings through click chemistry. Importantly, the dynamic surroundings exhibited negative charges under physiological conditions (pH 7.4). This property prevented degradation by anionic nucleases and structural disassembly induced by endogenous charged biological species. Consequently, the nanoparticles exhibited appreciable stealth function, effectively managing the first pass effect, leading to prolonged blood retention and improved bioavailabilities at targeted cells. Furthermore, the dynamic surroundings shifted towards relatively positive charges in the tumor microenvironment (pH 6.8). As a result, the nanoparticles were more likely to be taken up by tumors due to their electrostatic affinities towards polyanionic cytomembranes. Eventually, the internalized mRNA nanomedicine transformed responsive to the surrounding microenvironment into highly positive charges within acidic endolysosomes (pH 5.0), exerting explosive disruptive potencies on the endolysosomal structures, thus facilitating translocation of mRNA from the digestive endolysosomes into the targeted cytosol. Notably, the dynamic surroundings also reduced the immunogenicity of naked mRNA due to their stealthy properties and rapid endolysosomal translocation functions. In summary, our proposed unique triple-transformable dynamic surface chemistry provided an intriguing delivery scenario that overcomes sequential biological barriers, contributing to efficient expression of the encapsulated mRNA at targeted tumors.

Optimized PAR-2 RING dimerization mediates cooperative and selective membrane binding for robust cell polarity

Cell polarity networks are defined by quantitative features of their constituent feedback circuits, which must be tuned to enable robust and stable polarization, while also ensuring that networks remain responsive to dynamically changing cellular states and/or spatial cues during development. Using the PAR polarity network as a model, we demonstrate that these features are enabled by the dimerization of the polarity protein PAR-2 via its N-terminal RING domain. Combining theory and experiment, we show that dimer affinity is optimized to achieve dynamic, selective, and cooperative binding of PAR-2 to the plasma membrane during polarization. Reducing dimerization compromises positive feedback and robustness of polarization. Conversely, enhanced dimerization renders the network less responsive due to kinetic trapping of PAR-2 on internal membranes and reduced sensitivity of PAR-2 to the anterior polarity kinase, aPKC/PKC-3. Thus, our data reveal a key role for a dynamically oligomeric RING domain in optimizing interaction affinities to support a robust and responsive cell polarity network, and highlight how optimization of oligomerization kinetics can serve as a strategy for dynamic and cooperative intracellular targeting.

Antimicrobial peptides from plants and microorganisms for plant disease management

AbstractPlant disease control faces a lot of challenges due to its overdependence on chemicals that have strict restrictions and regulatory requirements. With the increase in drug‐resistant pathogens and continual crop losses due to disease outbreaks, much attention has been brought to a new set of emerging antibiotics called antimicrobial peptides (AMPs). AMPs are a group of multifunctional, short‐sequence peptides that are usually cationic in nature and found in all living organisms. They are part of the innate immune system of different organisms and exhibit a wide range of inhibitory effects on microorganisms, making them potential therapeutic factors efficacious as an alternate resource for plant disease management. AMPs interact with the anionic cell membrane of the pathogen and cause cell lysis or inhibit crucial intracellular targets. AMPs can be isolated from almost all life forms ranging from microbes to mammals. In addition to these sources, AMPs are also being synthesized using recombinant methods with the goal of overcoming the constraints of natural AMPs with regard to stability, activity and toxicity. Recent advancements have been made to develop transgenic plants expressing AMPs that has proved to perform better than the use of antibiotics. This review highlights the different kinds of AMPs produced by plants and microorganisms along with their mode of action, target pathogens, structural characteristics and advancements in this field, which include isolation methods, synthetic AMPs and transgenic plants expressing AMPs.

Exploring the Molecular Therapeutic Mechanisms of Gemcitabine through Quantitative Proteomics.

Gemcitabine (GEM) is widely employed in the treatment of various cancers, including pancreatic cancer. Despite their clinical success, challenges related to GEM resistance and toxicity persist. Therefore, a deeper understanding of its intracellular mechanisms and potential targets is urgently needed. In this study, through mass spectrometry analysis in data-dependent acquisition mode, we carried out quantitative proteomics (three independent replications) and thermal proteome profiling (TPP, two independent replications) on MIA PaCa-2 cells to explore the effects of GEM. Our proteomic analysis revealed that GEM led to the upregulation of the cell cycle and DNA replication proteins. Notably, we observed the upregulation of S-phase kinase-associated protein 2 (SKP2), a cell cycle and chemoresistance regulator. Combining SKP2 inhibition with GEM showed synergistic effects, suggesting SKP2 as a potential target for enhancing the GEM sensitivity. Through TPP, we pinpointed four potential GEM binding targets implicated in tumor development, including in breast and liver cancers, underscoring GEM's broad-spectrum antitumor capabilities. These findings provide valuable insights into GEM's molecular mechanisms and offer potential targets for improving treatment efficacy.

Association of Α-Klotho with regulation of Keap1/Nrf2/Interleukin-1 pathway and AMPA receptor trafficking in the brain of suicide victims.

Suicide is a significant public health challenge worldwide. Statistical data confirm a strong relationship between suicidal behavior and depressive disorders (DDs), but the molecular mechanisms of these diseases are still poorly understood. A growing body of research suggests that the Klotho-mediated pathway may be a novel intracellular target for the development of suicide-related disorders (including DDs). To verify this hypothesis, the link between α-Klotho levels, Nrf2-related inflammatory status (IL-1α, IL-1β, Keap1, NFκB p65), AMPA (GluA1, GluA2, p-S831-GluA1, p-S845-GluA1) receptor subunit trafficking and AMPK (AMPKα1/2; pT172-AMPKα1) signalling pathways in the brain of suicide victims as compared to controls were investigated. Commercially available enzyme-linked immunoassay (ELISA) and Western blot analysis were performed in the hippocampus (HP) and frontal cortex (FCx) of suicide victims and matched controls. Group differences were assessed using an unpaired Student's t-test. A statistically significant decrease in the level of α-Klotho (HP: p=0.001; FCx: p=0.012) with an increase in IL-1β (HP: p=0.0108) and IL-1α (FCx: p=0.009) concentrations were shown. These alterations were associated with increased Keap1 (FCx: p=0.023) and NF-κB-p65 (HP: p=0.039; FCx: p=0.013 nuclear fraction) protein levels. Furthermore, a significant reduction in p-S831-GluA1 (HP: p=0.029; FCx=0.002) and p-S845-GluA1 (HP: p=0.0012) proteins was observed. Similarly, the level of GluA2 (HP: p=0.011; FCx: p=0.002) and in p-T172-AMPKα1 (HP: p=0.0288; FCx: p=0.0338) protein were statistically decreased. Our findings demonstrate that a reduction in α-Klotho levels in brain structures related to mood disorders (HP, FCx) correlates with suicidal behavior. Moreover, our study provides novel insights into the molecular mechanisms underlying suicide-related disorders, highlighting the role of α-Klotho, Nrf2-related inflammatory status, AMPA receptor trafficking, and AMPK signaling pathways in the pathophysiology of suicidal behavior. These results may have implications for the development of targeted interventions for individuals at risk of suicide.

Oral Estrogen Receptor PROTAC Vepdegestrant (ARV-471) Is Highly Efficacious as Monotherapy and in Combination with CDK4/6 or PI3K/mTOR Pathway Inhibitors in Preclinical ER+ Breast Cancer Models.

Estrogen receptor (ER) alpha signaling is a known driver of ER-positive (ER+)/human epidermal growth factor receptor 2 negative (HER2-) breast cancer. Combining endocrine therapy (ET) such as fulvestrant with CDK4/6, mTOR, or PI3K inhibitors has become a central strategy in the treatment of ER+ advanced breast cancer. However, suboptimal ER inhibition and resistance resulting from the ESR1 mutation dictates that new therapies are needed. A medicinal chemistry campaign identified vepdegestrant (ARV-471), a selective, orally bioavailable, and potent small molecule PROteolysis-TArgeting Chimera (PROTAC) degrader of ER. We used biochemical and intracellular target engagement assays to demonstrate the mechanism of action of vepdegestrant, and ESR1 wild-type (WT) and mutant ER+ preclinical breast cancer models to demonstrate ER degradation-mediated tumor growth inhibition (TGI). Vepdegestrant induced ≥90% degradation of wild-type and mutant ER, inhibited ER-dependent breast cancer cell line proliferation in vitro, and achieved substantial TGI (87%-123%) in MCF7 orthotopic xenograft models, better than those of the ET agent fulvestrant (31%-80% TGI). In the hormone independent (HI) mutant ER Y537S patient-derived xenograft (PDX) breast cancer model ST941/HI, vepdegestrant achieved tumor regression and was similarly efficacious in the ST941/HI/PBR palbociclib-resistant model (102% TGI). Vepdegestrant-induced robust tumor regressions in combination with each of the CDK4/6 inhibitors palbociclib, abemaciclib, and ribociclib; the mTOR inhibitor everolimus; and the PI3K inhibitors alpelisib and inavolisib. Vepdegestrant achieved greater ER degradation in vivo compared with fulvestrant, which correlated with improved TGI, suggesting vepdegestrant could be a more effective backbone ET for patients with ER+/HER2- breast cancer.

Kinetic Behavior of Glutathione Transferases: Understanding Cellular Protection from Reactive Intermediates.

Glutathione transferases (GSTs) are the primary catalysts protecting from reactive electrophile attack. In this review, the quantitative levels and distribution of glutathione transferases in relation to physiological function are discussed. The catalytic properties (random sequential) tell us that these enzymes have evolved to intercept reactive intermediates. High concentrations of enzymes (up to several hundred micromolar) ensure efficient protection. Individual enzyme molecules, however, turn over only rarely (estimated as low as once daily). The protection of intracellular protein and DNA targets is linearly proportional to enzyme levels. Any lowering of enzyme concentration, or inhibition, would thus result in diminished protection. It is well established that GSTs also function as binding proteins, potentially resulting in enzyme inhibition. Here the relevance of ligand inhibition and catalytic mechanisms, such as negative co-operativity, is discussed. There is a lack of knowledge pertaining to relevant ligand levels in vivo, be they exogenous or endogenous (e.g., bile acids and bilirubin). The stoichiometry of active sites in GSTs is well established, cytosolic enzyme dimers have two sites. It is puzzling that a third of the site's reactivity is observed in trimeric microsomal glutathione transferases (MGSTs). From a physiological point of view, such sub-stoichiometric behavior would appear to be wasteful. Over the years, a substantial amount of detailed knowledge on the structure, distribution, and mechanism of purified GSTs has been gathered. We still lack knowledge on exact cell type distribution and levels in vivo however, especially in relation to ligand levels, which need to be determined. Such knowledge must be gathered in order to allow mathematical modeling to be employed in the future, to generate a holistic understanding of reactive intermediate protection.

Characterization of Argonaute-containing protein complexes in Leishmania-infected human macrophages.

The intracellular protozoan parasite Leishmania causes leishmaniasis in humans, leading to serious illness and death in tropical and subtropical areas worldwide. Unfortunately, due to the unavailability of approved vaccines for humans and the limited efficacy of available drugs, leishmaniasis is on the rise. A comprehensive understanding of host-pathogen interactions at the molecular level could pave the way to counter leishmaniasis. There is growing evidence that several intracellular pathogens target RNA interference (RNAi) pathways in host cells to facilitate their persistence. The core elements of the RNAi system are complexes of Argonaute (Ago) proteins with small non-coding RNAs, also known as RNA-induced silencing complexes (RISCs). Recently, we have shown that Leishmania modulates Ago1 protein of host macrophages for its survival. In this study, we biochemically characterize the Ago proteins' interactome in Leishmania-infected macrophages compared to non-infected cells. For this, a quantitative proteomic approach using stable isotope labelling by amino acids in cell culture (SILAC) was employed, followed by purification of host Ago-complexes using a short TNRC6 protein-derived peptide fused to glutathione S-transferase beads as an affinity matrix. Proteomic-based detailed biochemical analysis revealed Leishmania modulated host macrophage RISC composition during infection. This analysis identified 51 Ago-interacting proteins with a broad range of biological activities. Strikingly, Leishmania proteins were detected as part of host Ago-containing complexes in infected cells. Our results present the first report of comprehensive quantitative proteomics of Ago-containing complexes isolated from Leishmania-infected macrophages and suggest targeting the effector complex of host RNAi machinery. Additionally, these results expand knowledge of RISC in the context of host-pathogen interactions in parasitology in general.

Brain Delivery of Protein Therapeutics by Cell Matrix-Inspired Biomimetic Nanocarrier.

Protein therapeutics are anticipated to offer significant treatment options for central nervous system (CNS) diseases. However, the majority of proteins are unable to traverse the blood-brain barrier (BBB) and reach their CNS target sites. Inspired by the natural environment of active proteins, the cell matrix components hyaluronic acid (HA) and protamine (PRTM) are used to self-assemble with proteins to form a protein-loaded biomimetic core and then incorporated into ApoE3-reconstituted high-density lipoprotein (rHDL) to form a protein-loaded biomimetic nanocarrier (Protein-HA-PRTM-rHDL). This cell matrix-inspired biomimetic nanocarrier facilitates the penetration of protein therapeutics across the BBB and enables their access to intracellular target sites. Specifically, CAT-HA-PRTM-rHDL facilitates rapid intracellular delivery and release of catalase (CAT) via macropinocytosis-activated membrane fusion, resulting in improved spatial learning and memory in traumatic brain injury (TBI) model mice (significantly reduces the latency of TBI mice and doubles the number of crossing platforms), and enhances motor function and prolongs survival in amyotrophic lateral sclerosis (ALS) model mice (extended the median survival of ALS mice by more than 10 days). Collectively, this cell matrix-inspired nanoplatform enables the efficient CNS delivery of protein therapeutics and provides a novel approach for the treatment of CNS diseases.

Antibody-Loading of Biological Nanocarrier Vesicles Derived from Red-Blood-Cell Membranes.

Antibodies, disruptive potent therapeutic agents against pharmacological targets, face a barrier in crossing immune systems and cellular membranes. To overcome these, various strategies have been explored including shuttling via liposomes or biocamouflaged nanoparticles. Here, we demonstrate the feasibility of loading antibodies into exosome-mimetic nanovesicles derived from human red-blood-cell membranes, which can act as nanocarriers for intracellular delivery. Goat-antichicken antibodies are loaded into erythrocyte-derived nanovesicles, and their loading yields are characterized and compared with smaller dUTP-cargo molecules. Applying dual-color coincident fluorescence burst analyses, the loading yield of nanocarriers is rigorously profiled at the single-vesicle level, overcoming challenges due to size-heterogeneity and demonstrating a maximum antibody-loading yield of 38-41% at the optimal vesicle radius of 52 nm. The achieved average loading yields, amounting to 14% across the entire nanovesicle population, with more than two antibodies per loaded vesicle, are fully comparable to those obtained for the much smaller dUTP molecules loaded in the nanovesicles after additional exosome-spin-column purification. The results suggest a promising new avenue for therapeutic delivery of antibodies, potentially encompassing also intracellular targets and suitable for large-scale pharmacological applications, which relies on the exosome-mimetic properties, biocompatibility, and low-immunogenicity of bioengineered nanocarriers synthesized from human erythrocyte membranes.

Berberine inhibits intracellular Ca2+ signals in mouse pancreatic acinar cells through M3 muscarinic receptors: Novel target, mechanism, and implication

Berberine, a natural isoquinoline alkaloid, exhibits a variety of pharmacological effects, but the pharmacological targets and mechanisms remain elusive. Here, we report a novel finding that berberine inhibits acetylcholine (ACh)-induced intracellular Ca2+ oscillations, mediated through an inhibition of the muscarinic subtype 3 (M3) receptor. Patch-clamp recordings and confocal Ca2+ imaging were applied to acute dissociated pancreatic acinar cells prepared from CD1 mice to examine the effects of berberine on ACh-induced Ca2+ oscillations. Whole-cell patch-clamp recordings showed that berberine (from 0.1 to 10 µM) reduced ACh-induced Ca2+ oscillations in a concentration-dependent manner, and this inhibition also depended on ACh concentrations. The inhibitory effect of berberine neither occurred in intracellular targets nor extracellular cholecystokinin (CCK) receptors, chloride (Cl-) channels, and store-operated Ca2+ channels. Together, the results demonstrate that berberine directly inhibits the muscarinic M3 receptors, further confirmed by evidence of the interaction between berberine and M3 receptors in pancreatic acinar cells.

Triphenylphosphine-bonded coumaranone dyes realize dual color imaging of mitochondria and nucleoli

Probing intracellular organelles with fluorescent dyes offers opportunities to understand the structures and functions of these cellular compartments, which is attracting increasing interests. Normally, the design principle varies for different organelle targets as they possess distinct structural and functional profiles against each other. Therefore, developing a probe with dual intracellular targets is of great challenge. In this work, a new sort of donor–π–bridge–acceptor (D-π-A) type coumaranone dyes (CMO-1/2/3/4) have been prepared. Four fluorescent probes (TPP@CMO-1/2/3/4) were then synthesized by linking these coumaranone dyes with an amphiphilic cation triphenylphosphonium (TPP). Interestingly, both TPP@CMO-1 and TPP@CMO-2 exhibited dual color emission upon targeting to two different organelles, respectively. The green emission is well localized in mitochondria, while, the red emission realizes nucleoli imaging. RNA is the target of TPP@CMOs, which was confirmed by spectroscopic analysis and computational calculation. More importantly, the number and morphology changes of nucleoli under drug stress have been successfully evaluated using TPP@CMO-1.

Seeking Cells, Targeting Bacteria: A Cascade-Targeting Bacteria-Responsive Nanosystem for Combating Intracellular Bacterial Infections.

Intracellular bacteria pose a great challenge to antimicrobial therapy due to various physiological barriers at both cellular and bacterial levels, which impede drug penetration and intracellular targeting, thereby fostering antibiotic resistance and yielding suboptimal treatment outcomes. Herein, a cascade-target bacterial-responsive drug delivery nanosystem, MM@SPE NPs, comprising a macrophage membrane (MM) shell and a core of SPE NPs. SPE NPs consist of phenylboronic acid-grafted dendritic mesoporous silica nanoparticles (SP NPs) encapsulated with epigallocatechin-3-gallate (EGCG), a non-antibiotic antibacterial component, via pH-sensitive boronic ester bonds are introduced. Upon administration, MM@SPE NPs actively home in on infected macrophages due to the homologous targeting properties of the MM shell, which is subsequently disrupted during cellular endocytosis. Within the cellular environment, SPE NPs expose and spontaneously accumulate around intracellular bacteria through their bacteria-targeting phenylboronic acid groups. The acidic bacterial microenvironment further triggers the breakage of boronic ester bonds between SP NPs and EGCG, allowing the bacterial-responsive release of EGCG for localized intracellular antibacterial effects. The efficacy of MM@SPE NPs in precisely eliminating intracellular bacteria is validated in two rat models of intracellular bacterial infections. This cascade-targeting responsive system offers new solutions for treating intracellular bacterial infections while minimizing the risk of drug resistance.

A Conditionally Activated Cytosol-Penetrating Antibody for TME-Dependent Intracellular Cargo Delivery.

Currently, therapeutic and diagnostic applications of antibodies are primarily limited to cell surface-exposed and extracellular proteins. However, research has been conducted on cell-penetrating peptides (CPP), as well as cytosol-penetrating antibodies, to overcome these limitations. In this context, a heparin sulfate proteoglycan (HSPG)-binding antibody was serendipitously discovered, which eventually localizes to the cytosol of target cells. Functional characterization revealed that the tested antibody has beneficial cytosol-penetrating capabilities and can deliver cargo proteins (up to 70 kDa) to the cytosol. To achieve tumor-specific cell targeting and cargo delivery through conditional activation of the cell-penetrating antibody in the tumor microenvironment, a single-chain Fc fragment (scFv) and a VL domain were isolated as masking units. Several in vitro assays demonstrated that fusing the masking protein with a cleavable linker to the cell penetration antibody results in the inactivation of antibody cell binding and internalization. Removal of the mask via MMP-9 protease cleavage, a protease that is frequently overexpressed in the tumor microenvironment (TME), led to complete regeneration of binding and cytosol-penetrating capabilities. Masked and conditionally activated cytosol-penetrating antibodies have the potential to serve as a modular platform for delivering protein cargoes addressing intracellular targets in tumor cells.

Evaluation of Solubility and Membrane Permeability of Middle-Molecule Compounds Using Artificial Membranes and Living Cells

In contrast to small molecules, middle molecules present a promising therapeutic modality owing to their elevated specificity, minimal adverse effects, capacity to target protein-protein interactions, and, unlike antibody-based drugs, their suitability for oral administration and intracellular target engagement. Post-oral administration, the paramount considerations encompass solubility and membrane permeability during the initial phase until the drug attains systemic circulation. Furthermore, penetration of the cell membrane is essential to accessing intracellular targets. We evaluated the solubility and membrane permeability of 965 compounds sourced from middle molecule libraries affiliated with Hokkaido University, Kitasato University, and the University of Tokyo. To gauge membrane permeability, we employed both the parallel artificial membrane permeability assay (PAMPA) and Caco-2 cell monolayers. Notably, while membrane permeability in Caco-2 cells exhibited an approximate threefold increase in comparison to PAMPA measurements, certain compounds demonstrated permeability levels less than one-third of those observed in Caco-2 cells. Recognizing the potential involvement of efflux transporters expressed in Caco-2 cells in these variations, we conducted additional assessments involving directional transport in the presence of a transporter inhibitor. Our findings suggest that nearly 80% of these compounds serve as substrates for efflux transporters. Considering the relevance of intracellular targets, we shifted our focus from membrane permeation to intracellular uptake, conducting simulations tailored to assess cellular uptake.

Preparation and In vitro Evaluation of Folated Pluronic F87/TPGS Co-modified Liposomes for Targeted Delivery of Curcumin.

Using targeted liposomes to encapsulate and deliver drugs has become a hotspot in biomedical research. Folated Pluronic F87/D-α-tocopheryl polyethylene glycol 1000 succinate (TPGS) co-modified liposomes (FA-F87/TPGS-Lps) were fabricated for curcumin delivery, and intracellular targeting of liposomal curcumin was investigated. FA-F87 was synthesized and its structural characterization was conducted through dehydration condensation. Then, cur-FA-F87/TPGS-Lps were prepared via thin film dispersion method combined with DHPM technique, and their physicochemical properties and cytotoxicity were determined. Finally, the intracellular distribution of cur-FA-F87/TPGS-Lps was investigated using MCF-7 cells. Incorporation of TPGS in liposomes reduced their particle size, but increased the negative charge of the liposomes as well as their storage stability, and the encapsulation efficiency of curcumin was improved. While, modification of liposomes with FA increased their particle size, and had no impact on the encapsulation efficiency of curcumin in liposomes. Among all the liposomes (cur-F87-Lps, cur-FA-F87-Lps, cur-FA-F87/TPGS-Lps and cur-F87/TPGS-Lps), cur-FA-F87/TPGS-Lps showed highest cytotoxicity to MCF-7 cells. Moreover, cur-FA-F87/TPGS-Lps was found to deliver curcumin into the cytoplasm of MCF-7 cells. Folate-Pluronic F87/TPGS co-modified liposomes provide a novel strategy for drug loading and targeted delivery.

Berberine Alleviates Right Heart Dysfunction in MCT-Induced PAH Rats by Targeting Myocardial Mitochondrial Oxidative Phosphorylation

Background: Right ventricular failure (RVF) stands as a leading cause of mortality among pulmonary hypertension (PAH) patients. The underlying mechanisms contributing to Right Ventricular (RV) dysfunction remain enigmatic, with a conspicuous absence of effective treatments. RV functional deterioration is influenced by mitochondria oxidative phosphorylation and fatty acid oxidation. Berberine (BBR), a natural active compound extracted from Berberis vulgaris rhizomes, has shown promise for treating cardiovascular diseases due to its interactions with multiple intracellular targets. This study aimed to investigate the applicability and therapeutic effcacy of BBR in RVF and explore the mechanisms involved in its beneficial effects in monocrotaline (MCT)-induced PAH rats. Methods: Right ventricular hypertrophy (RVH) and RVF were induced via a single injection of MCT (60 mg/kg, ip) in Sprague-Dawley rats. BBR (4 mg/kg body weight per day) was administered through oral gavage. RVH and RVF were confirmed through transthoracic echocardiography (TTE). Transmission electron microscopy (TEM) was utilized to observe changes in cardiomyocyte and mitochondrial ultrastructure within the right ventricle tissue. Comparisons were made among Normal control (NC), RVH, RVH+BBR, RVF, and RVF+ BBR groups. RNA sequencing (RNA-seq) of right ventricle tissue was performed for both the NC (treated with normal saline, ip) and right ventricle dysfunction (RVH and RVF) groups. Gene Ontology and KEGG enrichment analyses were conducted using the R-package “Clusterprofiler.” RV transcriptome results were validated through qPCR and Western blot analysis. Results: Echocardiography and elevated ANP and BNP levels confirmed the presence of RVF following the development of MCT-induced PAH. TEM imaging revealed significant disruptions in mitochondrial ultrastructure, particularly in a subset of interfibrillar mitochondria, which exhibited swelling, disorganization, and reduced cristae density in RVH rats. These disruptions were even more pronounced in RVF rats. Transcriptomic profiles of RV tissue from RVH and RVF identified a total of 175 differentially expressed genes (DEGs). Gene Ontology and KEGG enrichment analyses pinpointed 38 genes associated with oxidative phosphorylation. qPCR demonstrated downregulation of mitochondrial oxidative phosphorylation-related genes (NDUFA2, NDUFA13, COX7B, COX6C, GSTK1, ECH1) in RVF. BBR ameliorated right heart function, leading to decreased RV internal diameter at end-diastole, a reduced RV hypertrophy index, and an increased RV fractional area change. This was accompanied by the recovery of ANP and BNP levels. Furthermore, after BBR application, the mitochondrial structure within RV tissue exhibited marked improvement. Lastly, Western blot analysis revealed that fatty acid oxidation-related proteins (Decr1, Acadm, Acacb, Hadh, Hadhb, Prkag3, Acaa2) were downregulated in right heart dysfunction, and BBR effectively mitigated the decline in oxidation-related protein levels in both RVH and RVF. Conclusion: In summary, our results underscore the critical role of BBR in preserving cardiac function from RVF in MCT-induced PAH rats. This preservation appears to occur through the upregulation of mitochondrial oxidative phosphorylation levels, ultimately enhancing energy metabolism within the mitochondria of RV cardiomyocytes in the right heart of PAH. This work is supported by Natural Science Foundation of Shandong Province(ZR2022MH052), China Postdoctoral Fund (NO.2021M691944) to XMY. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Opposing roles of STAT1 and STAT3 in COX-2 protein expression in human follicular dendritic cells

Abstract Prostaglandins (PGs) are the lipid mediators of inflammatory responses and acknowledged also as the mediators of immune inflammation. We have explored the role of PGs and their production mechanisms using follicular dendritic cell (FDC)-like cells. In the course of our recent study of the regulation mechanisms of PG production, we observed the involvement of JAK-STAT molecules in the IL-1β-induced PG production. In the current study, we aimed to elucidate the role of JAK and STAT proteins in the expression of COX-2 protein, the responsible enzyme for PG production. COX-2 protein level and PG production were assessed using immunoblotting and EIA, respectively. The intracellular target proteins were detected by a confocal microscopy. STAT1 knockdown resulted in an increase of COX-2 expression, while STAT3 knockdown led to a reduction in the COX-2 induction by IL-1β. IL-1β induced phosphorylation of both STAT1 and STAT3 at the S727 sites, rather than at Y701 or Y705. The phosphorylation and nuclear translocation of pSTAT1 and pSTAT3 were confirmed by a confocal microscopy. Based on these findings, we maintain that STAT1 may play an inhibitory role in COX-2 induction, while STAT3 acts as a promoter, via fine tuning of phosphorylation levels. The current study may enhance our understanding of the precise roles of these STAT proteins in COX-2 induction and PG production and suggests that these molecules could be potential targets for the treatment of immune inflammatory disorders.