RESUMEN
Endocytosis is a major bottleneck toward cytosolic delivery of nucleic acids, as the vast majority of nucleic acid drugs remain trapped within endosomes. Current trends to overcome endosomal entrapment and subsequent degradation provide varied success; however, active delivery agents such as cell-penetrating peptides have emerged as a prominent strategy to improve cytosolic delivery. Yet, these membrane-active agents have poor selectivity for endosomal membranes, leading to toxicity. A hallmark of endosomes is their acidic environment, which aids in degradation of foreign materials. Here, we develop a pH-triggered spherical nucleic acid that provides smart antisense oligonucleotide (ASO) release upon endosomal acidification and selective membrane disruption, termed DNA EndosomaL Escape Vehicle Response (DELVR). We anchor i-Motif DNA to a nanoparticle (AuNP), where the complement strand contains both an ASO sequence and a functionalized endosomal escape peptide (EEP). By orienting the EEP toward the AuNP core, the EEP is inactive until it is released through acidification-induced i-Motif folding. In this study, we characterize a small library of i-Motif duplexes to develop a structure-switching nucleic acid sequence triggered by endosomal acidification. We evaluate antisense efficacy using HIF1a, a hypoxic indicator upregulated in many cancers, and demonstrate dose-dependent activity through RT-qPCR. We show that DELVR significantly improves ASO efficacy in vitro. Finally, we use fluorescence lifetime imaging and activity measurement to show that DELVR benefits synergistically from nuclease- and pH-driven release strategies with increased ASO endosomal escape efficiency. Overall, this study develops a modular platform that improves the cytosolic delivery of nucleic acid therapeutics and offers key insights for overcoming intracellular barriers.
Asunto(s)
Ácidos Nucleicos , Ácidos Nucleicos/metabolismo , Endosomas/química , Endocitosis/fisiología , Membranas Intracelulares , ADN/metabolismoRESUMEN
In this perspective, we outline a new opportunity for exploiting nanoparticle delivery of antagonists to target G-protein coupled receptors localized in intracellular compartments. We discuss the specific example of antagonizing endosomal receptors involved in pain to develop long-lasting analgesics but also outline the broader application potential of this delivery approach. We discuss the materials used to target endosomal receptors and indicate the design requirements for future successful applications.
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Endosomas , Nanomedicina , Animales , Humanos , Endosomas/química , Nanomedicina/métodos , Nanopartículas/química , Polímeros/química , Concentración de Iones de Hidrógeno , Sistemas de Liberación de MedicamentosRESUMEN
Cell-penetrating peptides (CPPs) are prominent scaffolds for drug developments and related research, particularly the endocytic delivery of biomacromolecules. Effective cargo release from endosomes prior to lysosomal degradation is a crucial step, where the rational design and selection of CPPs remains a challenge and calls for deeper mechanistic understandings. Here, we have investigated a strategy of designing CPPs that selectively disrupt endosomal membranes based on bacterial membrane targeting sequences (MTSs). Six synthesized MTS peptides all exhibit cell-penetrating abilities, among which two d-peptides (d-EcMTS and d-TpMTS) are able to escape from endosomes and localize at ER after entering the cell. The utility of this strategy has been demonstrated by the intracellular delivery of green fluorescent protein (GFP). Together, these results suggest that the large pool of bacterial MTSs may be a rich source for the development of novel CPPs.
Asunto(s)
Péptidos de Penetración Celular , Péptidos de Penetración Celular/química , Endosomas/química , Endosomas/metabolismoRESUMEN
Pore-forming toxins (PFTs) are important virulence factors produced by many pathogenic bacteria. Here, we show that the Vibrio cholerae toxin MakA is a novel cholesterol-binding PFT that induces non-canonical autophagy in a pH-dependent manner. MakA specifically binds to cholesterol on the membrane at pH < 7. Cholesterol-binding leads to oligomerization of MakA on the membrane and pore formation at pH 5.5. Unlike other cholesterol-dependent cytolysins (CDCs) which bind cholesterol through a conserved cholesterol-binding motif (Thr-Leu pair), MakA contains an Ile-Ile pair that is essential for MakA-cholesterol interaction. Following internalization, endosomal acidification triggers MakA pore-assembly followed by ESCRT-mediated membrane repair and V-ATPase-dependent unconventional LC3 lipidation on the damaged endolysosomal membranes. These findings characterize a new cholesterol-binding toxin that forms pores in a pH-dependent manner and reveals the molecular mechanism of host autophagy manipulation.
Asunto(s)
Autofagia , Proteínas Bacterianas , Colesterol , Citotoxinas , Vibrio cholerae , Factores de Virulencia , Adenosina Trifosfatasas/metabolismo , Secuencias de Aminoácidos , Autofagia/efectos de los fármacos , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Colesterol/metabolismo , Citotoxinas/metabolismo , Citotoxinas/farmacología , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Endosomas/química , Endosomas/metabolismo , Concentración de Iones de Hidrógeno , Lisosomas/química , Lisosomas/metabolismo , Unión Proteica , Vibrio cholerae/química , Vibrio cholerae/metabolismo , Factores de Virulencia/química , Factores de Virulencia/metabolismoRESUMEN
For efficient delivery of messenger (m)RNA, delivery carriers need two major functions: protecting mRNA from nucleases and translocating mRNA from endolysosomes to the cytoplasm. Herein, these two complementary functionalities are integrated into a single polyplex by fine-tuning the catiomer chemical structure and incorporating the endosomal escape modality. The effect of the methylene spacer length on the catiomer side chain is evaluated by comparing poly(l-lysine) (PLL) with a tetramethylene spacer and poly(L-ornithine) (PLO) with a trimethylene spacer. Noteworthily, the nuclease stability of the mRNA/catiomer polyplexes is largely affected by the difference in one methylene group, with PLO/mRNA polyplex showing enhanced stability compared to PLL/mRNA polyplex. To introduce the endosomal escape function, the PLO/mRNA polyplex is wrapped with a charge-conversion polymer (CCP), which is negatively charged at extracellular pH but turns positive at endosomal acidic pH to disrupt the endosomal membrane. Compared to the parent PLO/mRNA polyplex, CCP facilitated the endosomal escape of the polyplex in cultured cells to improve the protein expression efficiency from mRNA by approximately 80-fold. Collectively, this system synergizes the protective effect of PLO against nucleases and the endosomal escape capability of CCP in mRNA delivery.
Asunto(s)
Endosomas , Polímeros , Endosomas/química , Endosomas/metabolismo , Ornitina/análisis , Ornitina/metabolismo , Polímeros/química , ARN Mensajero , TransfecciónRESUMEN
Exploring the cross-talk between the immune system and advanced biomaterials to treat SARS-CoV-2 infection is a promising strategy. Here, we show that ACE2-overexpressing A549 cell-derived microparticles (AO-MPs) are a potential therapeutic agent against SARS-CoV-2 infection. Intranasally administered AO-MPs dexterously navigate the anatomical and biological features of the lungs to enter the alveoli and are taken up by alveolar macrophages (AMs). Then, AO-MPs increase the endosomal pH but decrease the lysosomal pH in AMs, thus escorting bound SARS-CoV-2 from phago-endosomes to lysosomes for degradation. This pH regulation is attributable to oxidized cholesterol, which is enriched in AO-MPs and translocated to endosomal membranes, thus interfering with proton pumps and impairing endosomal acidification. In addition to promoting viral degradation, AO-MPs also inhibit the proinflammatory phenotype of AMs, leading to increased treatment efficacy in a SARS-CoV-2-infected mouse model without side effects. These findings highlight the potential use of AO-MPs to treat SARS-CoV-2-infected patients and showcase the feasibility of MP therapies for combatting emerging respiratory viruses in the future.
Asunto(s)
Enzima Convertidora de Angiotensina 2/administración & dosificación , Enzima Convertidora de Angiotensina 2/metabolismo , COVID-19/metabolismo , COVID-19/terapia , Tratamiento Basado en Trasplante de Células y Tejidos/métodos , Micropartículas Derivadas de Células/metabolismo , Colesterol/metabolismo , Endosomas/química , Macrófagos Alveolares/metabolismo , SARS-CoV-2/metabolismo , Células A549 , Enzima Convertidora de Angiotensina 2/genética , Animales , COVID-19/virología , Chlorocebus aethiops , Modelos Animales de Enfermedad , Femenino , Humanos , Concentración de Iones de Hidrógeno , Lisosomas/química , Ratones , Ratones Endogámicos ICR , Ratones Transgénicos , Oxidación-Reducción , Células RAW 264.7 , Resultado del Tratamiento , Células VeroRESUMEN
Rapid and efficient isolation of intact lysosomes is necessary to study their functions and metabolites by proteomic analysis. We developed a swift and robust nanoparticle-based magnetic separation method in which magnetic-plasmonic hybrid nanoparticles (MPNPs) conjugated with amino dextran (aDxt) were targeted to the lumen of lysosomes via the endocytosis pathway. For well-directed magnetic separation of the lysosomes, it is important to trace the intracellular trafficking of the aDxt-conjugated MPNPs (aDxt-MPNPs) in the endocytosis pathway. Therefore, we analyzed the intracellular transport process of the aDxt-MPNPs by investigating the time-dependent colocalization of plasmonic scattering of aDxt-MPNPs and immunostained marker proteins of organelles using the threshold Manders' colocalization coefficient (Rt). Detailed analysis of time variations of Rt for early and late endosomes and lysosomes allowed us to derive the transport kinetics of aDxt-MPNPs in a cell. After confirming the incubation time required for sufficient accumulation of aDxt-MPNPs in lysosomes, the lysosomes were magnetically isolated as intact as possible. By varying the elapsed time from homogenization to complete isolation of lysosomes (tdelay) and temperature (T), the influences of tdelay and T on the protein composition of the lysosomes were investigated by polyacrylamide gel electrophoresis and amino acid analysis. We found that the intactness of lysosomes could become impaired quite quickly, and to isolate lysosomes as intact as possible with high purity, tdelay = 30 min and T = 4 °C were optimal settings.
Asunto(s)
Endocitosis , Nanopartículas , Proteómica , Lisosomas/metabolismo , Endosomas/química , Fenómenos MagnéticosRESUMEN
Although cell-penetrating peptides such as the HIV-derived TAT peptide have been used as tools for the intracellular delivery of therapeutic peptides and proteins, a problem persists: the endosomal escape efficiency is low. Previously, we found that the fusogenic peptide S19, derived from the human protein syncytin-1, enhance the endosomal escape efficiency of proteins that incorporated by endocytosis via TAT. In this study, we first performed Ala-scanning mutagenesis of S19, and found that all Ile, Val, Leu and Phe with high ß-sheet forming propensities in S19 are important for the intracellular uptake of S19-TAT-fused proteins. In a secondary structure analysis of the mutated S19-TAT peptides in the presence of liposomes mimicking late endosomes (LEs), the CD spectra of V3A and I4A mutants with low uptake activity showed the appearance of an α-helix structure, whereas the mutant G5A retained both the uptake activity and the ß-structure. In addition, we investigated the appropriate linking position and order of the S19 and TAT peptides to a cargo protein including an apoptosis-induced peptide and found that both the previous C-terminal S19-TAT tag and the N-terminal TAT-S19 tag promote the cytoplasmic delivery of the fusion protein. These results and previous results suggest that the interaction of TAT with the LE membrane causes a structural change in S19 from a random coil to a ß-strand and that the subsequent parallel ß-sheet formation between two S19 peptides may promote adjacent TAT dimerization, resulting in endosomal escape from the LE membrane.
Asunto(s)
Membrana Celular/metabolismo , Productos del Gen env/metabolismo , Productos del Gen tat/metabolismo , Péptidos/metabolismo , Plásmidos/metabolismo , Proteínas Gestacionales/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Sustitución de Aminoácidos , Línea Celular Tumoral , Permeabilidad de la Membrana Celular , Endosomas/química , Endosomas/metabolismo , Expresión Génica , Productos del Gen env/genética , Productos del Gen tat/genética , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HeLa , Humanos , Liposomas/química , Liposomas/metabolismo , Imagen Óptica , Péptidos/genética , Plásmidos/química , Proteínas Gestacionales/genética , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Transporte de Proteínas , Proteínas Recombinantes de Fusión/genética , Transducción GenéticaRESUMEN
All nanoparticles have the potential to revolutionize the delivery of therapeutic cargo such as peptides, proteins, and RNA. However, effective cytosolic delivery of cargo from nanoparticles represents a significant challenge in the design of more efficient drug delivery vehicles. Recently, research has centered on designing nanoparticles with the capacity to escape endosomes by responding to biological stimuli such as changes in pH, which occur when nanoparticles are internalized into the endo-/lysosomal pathway. Current endosomal escape assays rely on indirect measurements and yield little quantitative information, which hinders the design of more efficient drug delivery vehicles. Therefore, we adapted the highly sensitive split luciferase endosomal escape quantification (SLEEQ) assay to better understand nanoparticle-induced endosomal escape. We applied SLEEQ to evaluate the endosomal escape behavior of two pH-responsive nanoparticles: the first with a poly(2-diisopropylamino ethyl methacrylate) (PDPAEMA) core and the second with 1:1 ratio of poly(2-diethylamino ethyl methacrylate) (PDEAEMA) and PDPAEMA. SLEEQ directly measured the cytosolic delivery and showed that engineering the nanoparticle disassembly pH could improve the endosomal escape efficiency by fivefold. SLEEQ is a versatile assay that can be used for a wide range of nanomaterials and will improve the development of drug delivery vehicles in the future.
Asunto(s)
Materiales Biocompatibles/metabolismo , Endosomas/metabolismo , Luciferasas/metabolismo , Nanopartículas/metabolismo , Materiales Biocompatibles/química , Endosomas/química , Concentración de Iones de Hidrógeno , Luciferasas/química , Ensayo de Materiales , Nanopartículas/químicaRESUMEN
Rhinoviruses (RVs) cause recurrent infections of the nasal and pulmonary tracts, life-threatening conditions in chronic respiratory illness patients, predisposition of children to asthmatic exacerbation, and large economic cost. RVs are difficult to treat. They rapidly evolve resistance and are genetically diverse. Here, we provide insight into RV drug resistance mechanisms against chemical compounds neutralizing low pH in endolysosomes. Serial passaging of RV-A16 in the presence of the vacuolar proton ATPase inhibitor bafilomycin A1 (BafA1) or the endolysosomotropic agent ammonium chloride (NH4Cl) promoted the emergence of resistant virus populations. We found two reproducible point mutations in viral proteins 1 and 3 (VP1 and VP3), A2526G (serine 66 to asparagine [S66N]), and G2274U (cysteine 220 to phenylalanine [C220F]), respectively. Both mutations conferred cross-resistance to BafA1, NH4Cl, and the protonophore niclosamide, as identified by massive parallel sequencing and reverse genetics, but not the double mutation, which we could not rescue. Both VP1-S66 and VP3-C220 locate at the interprotomeric face, and their mutations increase the sensitivity of virions to low pH, elevated temperature, and soluble intercellular adhesion molecule 1 receptor. These results indicate that the ability of RV to uncoat at low endosomal pH confers virion resistance to extracellular stress. The data endorse endosomal acidification inhibitors as a viable strategy against RVs, especially if inhibitors are directly applied to the airways. IMPORTANCE Rhinoviruses (RVs) are the predominant agents causing the common cold. Anti-RV drugs and vaccines are not available, largely due to rapid evolutionary adaptation of RVs giving rise to resistant mutants and an immense diversity of antigens in more than 160 different RV types. In this study, we obtained insight into the cell biology of RVs by harnessing the ability of RVs to evolve resistance against host-targeting small chemical compounds neutralizing endosomal pH, an important cue for uncoating of normal RVs. We show that RVs grown in cells treated with inhibitors of endolysosomal acidification evolved capsid mutations yielding reduced virion stability against elevated temperature, low pH, and incubation with recombinant soluble receptor fragments. This fitness cost makes it unlikely that RV mutants adapted to neutral pH become prevalent in nature. The data support the concept of host-directed drug development against respiratory viruses in general, notably at low risk of gain-of-function mutations.
Asunto(s)
Cápside/química , Mutación/efectos de los fármacos , Rhinovirus/fisiología , Desencapsidación Viral/fisiología , Antivirales/farmacología , Cápside/efectos de los fármacos , Proteínas de la Cápside/genética , Proteínas de la Cápside/metabolismo , Farmacorresistencia Viral/efectos de los fármacos , Farmacorresistencia Viral/genética , Endosomas/química , Endosomas/efectos de los fármacos , Endosomas/metabolismo , Células HeLa , Humanos , Concentración de Iones de Hidrógeno , Molécula 1 de Adhesión Intercelular/metabolismo , Conformación Proteica , Rhinovirus/química , Rhinovirus/efectos de los fármacos , Rhinovirus/genética , Virión/química , Virión/genética , Virión/metabolismo , Internalización del Virus/efectos de los fármacos , Desencapsidación Viral/efectos de los fármacos , Desencapsidación Viral/genéticaRESUMEN
Bluetongue virus (BTV) is a non-enveloped virus and causes substantial morbidity and mortality in ruminants such as sheep. Fashioning a receptor-binding protein (VP2) and a membrane penetration protein (VP5) on the surface, BTV releases its genome-containing core (VP3 and VP7) into the host cell cytosol after perforation of the endosomal membrane. Unlike enveloped ones, the entry mechanisms of non-enveloped viruses into host cells remain poorly understood. Here we applied single-particle cryo-electron microscopy, cryo-electron tomography and structure-guided functional assays to characterize intermediate states of BTV cell entry in endosomes. Four structures of BTV at the resolution range of 3.4-3.9 Å show the different stages of structural rearrangement of capsid proteins on exposure to low pH, including conformational changes of VP5, stepwise detachment of VP2 and a small shift of VP7. In detail, sensing of the low-pH condition by the VP5 anchor domain triggers three major VP5 actions: projecting the hidden dagger domain, converting a surface loop to a protonated ß-hairpin that anchors VP5 to the core and stepwise refolding of the unfurling domains into a six-helix stalk. Cryo-electron tomography structures of BTV interacting with liposomes show a length decrease of the VP5 stalk from 19.5 to 15.5 nm after its insertion into the membrane. Our structures, functional assays and structure-guided mutagenesis experiments combined indicate that this stalk, along with dagger domain and the WHXL motif, creates a single pore through the endosomal membrane that enables the viral core to enter the cytosol. Our study unveils the detailed mechanisms of BTV membrane penetration and showcases general methods to study cell entry of other non-enveloped viruses.
Asunto(s)
Virus de la Lengua Azul/metabolismo , Lengua Azul/virología , Proteínas de la Cápside/metabolismo , Endosomas/virología , Animales , Virus de la Lengua Azul/química , Virus de la Lengua Azul/genética , Virus de la Lengua Azul/ultraestructura , Proteínas de la Cápside/química , Proteínas de la Cápside/genética , Microscopía por Crioelectrón , Endosomas/química , Concentración de Iones de Hidrógeno , Modelos Moleculares , Ovinos , Enfermedades de las Ovejas/virología , Internalización del VirusRESUMEN
Human apoptosis-linked gene-2 interacting protein X (ALIX), a versatile adapter protein, regulates essential cellular processes by shuttling between late endosomal membranes and the cytosol, determined by its interactions with Src kinase. Here, we investigate the molecular basis of these transitions and the effects of tyrosine phosphorylation on the interplay between structure, assembly, and intramolecular and intermolecular interactions of ALIX. As evidenced by transmission electron microscopy, fluorescence and circular dichroism spectroscopy, the proline-rich domain of ALIX, which encodes binding epitopes of multiple cellular partners, formed rope-like ß-sheet-rich reversible amyloid fibrils that dissolved upon Src-mediated phosphorylation and were restored on protein-tyrosine phosphatase 1B-mediated dephosphorylation of its conserved tyrosine residues. Analyses of the Bro1 domain of ALIX by solution NMR spectroscopy elucidated the conformational changes originating from its phosphorylation by Src and established that Bro1 binds to hyperphosphorylated proline-rich domain and to analogs of late endosomal membranes via its highly basic surface. These results uncover the autoinhibition mechanism that relocates ALIX to the cytosol and the diverse roles played by tyrosine phosphorylation in cellular and membrane functions of ALIX.
Asunto(s)
Amiloide , Proteínas de Unión al Calcio , Proteínas de Ciclo Celular , Complejos de Clasificación Endosomal Requeridos para el Transporte , Endosomas , Membranas Intracelulares , Amiloide/química , Amiloide/metabolismo , Proteínas de Unión al Calcio/química , Proteínas de Unión al Calcio/metabolismo , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/química , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Endosomas/química , Endosomas/metabolismo , Humanos , Membranas Intracelulares/química , Membranas Intracelulares/metabolismo , Resonancia Magnética Nuclear Biomolecular , Fosforilación , Dominios Proteicos , Relación Estructura-Actividad , TirosinaRESUMEN
Endosomal escape continues to be a limiting factor in the therapeutic use of nanomaterials. Assays to visualize endosomal escape often do not decouple the endosomal/lysosomal disruption from the release of payload into the cytosol. Here, we discuss three approaches to directly probe endosomal/lysosomal rupture: calcein dye dilution, lysosome size quantification and endosome/lysosome membrane integrity visualized with a genetically engineered cell line. We apply the three assays to endosomes/lysosomes ruptured via osmotic pressure and photochemical internalization.
Asunto(s)
Endosomas/química , Citosol/química , Fluoresceínas/química , Humanos , Lisosomas/química , Presión Osmótica , Procesos FotoquímicosRESUMEN
Niemann-Pick type C1 (NPC1) receptor is an endosomal membrane protein that regulates intracellular cholesterol traffic. This protein has been shown to play an important role for several viruses. It has been reported that SARS-CoV-2 enters the cell through plasma membrane fusion and/or endosomal entry upon availability of proteases. However, the whole process is not fully understood yet and additional viral/host factors might be required for viral fusion and subsequent viral replication. Here, we report a novel interaction between the SARS-CoV-2 nucleoprotein (N) and the cholesterol transporter NPC1. Furthermore, we have found that some compounds reported to interact with NPC1, carbazole SC816 and sulfides SC198 and SC073, were able to reduce SARS-CoV-2 viral infection with a good selectivity index in human cell infection models. These findings suggest the importance of NPC1 for SARS-CoV-2 viral infection and a new possible potential therapeutic target to fight against COVID-19.
Asunto(s)
Transporte Biológico , Tratamiento Farmacológico de COVID-19 , Endosomas/virología , Proteína Niemann-Pick C1/análisis , SARS-CoV-2/fisiología , Animales , Carbazoles/farmacología , Chlorocebus aethiops , Endosomas/química , Células HEK293 , Humanos , Péptidos y Proteínas de Señalización Intracelular , Fusión de Membrana , Células Vero , Replicación ViralRESUMEN
In this study, we report pH-responsive metal-based biopolymer nanoparticles (NPs) for tumor-specific chemotherapy. Here, aminated hyaluronic acid (aHA) coupled with 2,3-dimethylmaleic anhydride (DMA, as a pH-responsive moiety) (aHA-DMA) was electrostatically complexed with ferrous chloride tetrahydrate (FeCl2/4H2O, as a chelating metal) and doxorubicin (DOX, as an antitumor drug model), producing DOX-loaded Fe-based hyaluronate nanoparticles (DOX@aHA-DMA/Fe NPs). Importantly, the DOX@aHA-DMA/Fe NPs improved tumor cellular uptake due to HA-mediated endocytosis for tumor cells overexpressing CD44 receptors. As a result, the average fluorescent DOX intensity observed in MDA-MB-231 cells (with CD44 receptors) was ~7.9 × 102 (DOX@HA/Fe NPs, without DMA), ~8.1 × 102 (DOX@aHA-DMA0.36/Fe NPs), and ~9.3 × 102 (DOX@aHA-DMA0.60/Fe NPs). Furthermore, the DOX@aHA-DMA/Fe NPs were destabilized due to ionic repulsion between Fe2+ and DMA-detached aHA (i.e., positively charged free aHA) in the acidic environment of tumor cells. This event accelerated the release of DOX from the destabilized NPs. Our results suggest that these NPs can be promising tumor-targeting drug carriers responding to acidic endosomal pH.
Asunto(s)
Doxorrubicina/química , Endosomas/química , Compuestos Ferrosos/química , Ácido Hialurónico/química , Nanopartículas/química , Animales , Antineoplásicos/química , Línea Celular Tumoral , Portadores de Fármacos/química , Sistemas de Liberación de Medicamentos/métodos , Endocitosis/efectos de los fármacos , Femenino , Humanos , Receptores de Hialuranos/metabolismo , Concentración de Iones de Hidrógeno , Ratones , Ratones Endogámicos BALB CRESUMEN
Toll-like receptors (TLRs) are members of a large family of evolutionarily conserved pattern recognition receptors (PRRs), which serve as key components of the innate immune system by playing a pivotal role in sensing "nonself" ligands. Endosomal TLRs (TLR3, TLR7, TLR8, and TLR9) can recognize pathogen-derived nucleic acid and initiate an innate immune response because they react against both self- and non-self-origin nucleic acid molecules. Accordingly, both receptor agonists and antagonists are potentially useful in disparate clinical contexts and thus are globally sought after. Recent research has revealed that agonists and antagonists share an overlapping binding region. This Perspective highlights rational medicinal chemistry approaches to elucidate the structural attributes of small molecules capable of agonism or antagonism or of elegantly switching between the two. The structural evolution of different chemotypes can provide the framework for the future development of endosomal TLR agonists and antagonists.
Asunto(s)
Compuestos Heterocíclicos/química , Receptores Toll-Like/agonistas , Receptores Toll-Like/antagonistas & inhibidores , Animales , Endosomas/química , Células HEK293 , Compuestos Heterocíclicos/metabolismo , Compuestos Heterocíclicos/farmacología , Humanos , Ratones , Simulación del Acoplamiento Molecular , Estructura Molecular , Relación Estructura-Actividad , Receptores Toll-Like/metabolismoRESUMEN
To enable the release of the encapsulated nucleic acids into the cytosol of targeted cells, the interaction of lipid nanoparticles (LNPs) with endosomes is critical. We investigated changes in the physicochemical properties of LNPs containing ionizable cationic lipids that were induced by acidic pH, which reflects the conditions in the maturation of endosomes. We prepared a LNP containing an ionizable cationic lipid. The laurdan generalized polarization values, which are related to the hydration degree of the lipid membrane interface and are often used as an indicator of membrane packing, decreased with a decrease in pH value, showing that the membrane packing was decreased under acidic conditions. Furthermore, the pH-induced variation increased with an increasing percentage of ionizable cationic lipids in the LNPs. These results indicated that electrostatic repulsion between lipid molecules at acidic pH decreased the packing density of the lipids in the LNP membrane. Reducing the order of lipids could be a trigger to form a non-bilayer structure and allow fusion of the LNPs with the membrane of maturing endosomes in an acidic environment. The LNPs were used to incorporate and transport small interfering RNA (siRNA) into cells for knockdown of the expression of ß-galactosidase. The knockdown efficiency of siRNA encapsulated in LNPs tended to increase with the ratio of KC2. These results, which demonstrate the underlying phenomena for the fusion of membranes, will help clarify the mechanism of the release of encapsulated nucleic acids.
Asunto(s)
Lípidos/química , Lípidos de la Membrana/química , Nanopartículas/química , ARN Interferente Pequeño/química , Ácidos/farmacología , Endosomas/química , Técnicas de Transferencia de Gen , Humanos , Concentración de Iones de Hidrógeno , Lípidos/farmacología , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/farmacologíaRESUMEN
Despite the introduction of antiretroviral (ARV) therapy (ART), approximately 30-50% of people living with human immunodeficiency virus-1 (HIV-1) will develop a spectrum of measurable neurocognitive dysfunction, collectively called HIV-associated neurocognitive disorder (HAND). While the clinical manifestations of HAND have changed with the advent of ART, certain pathological features have endured, including white matter alterations and dysfunction. The persistence of white matter alterations in the post-ART era suggests that ARV drugs themselves may contribute to HAND pathology. Our group has previously demonstrated that two ARV compounds from the protease inhibitor (PI) class, ritonavir and lopinavir, inhibit oligodendrocyte maturation and myelin protein production. We hypothesized that other members of the PI class, saquinavir and darunavir, could also negatively impact oligodendrocyte differentiation. Here we demonstrate that treating primary rat oligodendrocyte precursor cells with therapeutically relevant concentrations of either ARV drug results in a concentration-dependent inhibition of oligodendrocyte maturation in vitro. Furthermore, we show that acidifying endolysosomal pH via a mucolipin transient receptor potential channel 1 (TRPML1) agonist provides protection against saquinavir- and darunavir-induced inhibition of oligodendrocyte maturation. Moreover, our findings suggest, for the first time, an imperative role of proper endolysosomal pH in regulating OL differentation, and that therapeutic targeting of endolysosomes may provide protection against ARV-induced oligodendrocyte dysregulation. Graphical Abstract Treatment of primary rat oligodendrocyte precursor cells with therapeutically relevant concentrations of either antiretroviral compound of the protease inhibitor class, darunavir or saquinavir, results in a concentration-dependent inhibition of oligodendrocyte maturation in vitro. Additionally, in darunavir or saquinavir-treated cultures we observed a concentration-dependent decrease in the number of acidic lysosomes, via immunostaining with LysoTracker Red, compared with vehicle-treated cultures. Finally, we showed that acidifying endolysosomal pH via a mucolipin transient receptor potential channel 1 (TRPML1) agonist provides protection against saquinavir- or darunavir-induced inhibition of oligodendrocyte maturation. Our findings suggest, for the first time, a critical role of proper endolysosomal pH in regulating OL differentation, and that therapeutic targeting of endolysosomes may provide protection against antiretroviral-induced oligodendrocyte dysregulation.
Asunto(s)
Darunavir/farmacología , Endosomas/efectos de los fármacos , Inhibidores de la Proteasa del VIH/farmacología , Lisosomas/efectos de los fármacos , Oligodendroglía/efectos de los fármacos , Saquinavir/farmacología , Animales , Apoptosis/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Darunavir/toxicidad , Depresión Química , Relación Dosis-Respuesta a Droga , Endosomas/química , Inhibidores de la Proteasa del VIH/toxicidad , Concentración de Iones de Hidrógeno , Lisosomas/química , Proteínas de la Mielina/biosíntesis , Estrés Oxidativo , Ftalimidas/farmacología , Quinolinas/farmacología , Ratas , Ratas Sprague-Dawley , Saquinavir/toxicidad , Canales de Potencial de Receptor Transitorio/agonistasRESUMEN
Antiretroviral therapeutics (ART) have effectively increased the long-term survival of HIV-1 infected individuals. However, the prevalence of HIV-1 associated neurocognitive disorders (HAND) has increased and so too have clinical manifestations and pathological features of Alzheimer's disease (AD) in people living with HIV-1/AIDS. Although underlying mechanisms are not clear, chronic exposure to ART drugs has been implicated in the development of AD-like symptoms and pathology. ART drugs are categorized according to their mechanism of action in controlling HIV-1 levels. All ART drugs are organic compounds that can be classified as being either weak acids or weak bases, and these physicochemical properties may be of central importance to ART drug-induced AD-like pathology because weak bases accumulate in endolysosomes, weak bases can de-acidify endolysosomes where amyloidogenesis occurs, and endolysosome de-acidification increases amyloid beta (Aß) protein production and decreases Aß degradation. Here, we investigated the effects of ART drugs on endolysosome pH and Aß levels in rat primary cultured neurons. ART drugs that de-acidified endolysosomes increased Aß levels, whereas those that acidified endolysosomes decreased Aß levels. Acidification of endolysosomes with the mucolipin transient receptor potential (TRPML) channel agonist ML-SA1 blocked ART drug-induced increases in Aß levels. Further, ART drug-induced endolysosome de-acidification increased endolysosome sizes; effects that were blocked by ML-SA1-induced endolysosome acidification. These results suggest that ART drug-induced endolysosome de-acidification plays an important role in ART drug-induced amyloidogenesis and that endolysosome acidification might attenuate AD-like pathology in HIV-1 positive people taking ART drugs that de-acidify endolysosomes. Graphical Abstract.
Asunto(s)
Amiloide/biosíntesis , Amiloidosis/inducido químicamente , Fármacos Anti-VIH/farmacología , Endosomas/efectos de los fármacos , Lisosomas/efectos de los fármacos , Amiloide/genética , Péptidos beta-Amiloides/metabolismo , Animales , Fármacos Anti-VIH/uso terapéutico , Línea Celular Tumoral , Células Cultivadas , Cloroquina/farmacología , Endosomas/química , Hipocampo/citología , Humanos , Concentración de Iones de Hidrógeno , Microscopía Intravital , Lisosomas/química , Neuroblastoma/patología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Proteolisis/efectos de los fármacos , Ratas , Ratas Sprague-DawleyRESUMEN
Lipid nanoparticles (LNPs) represent the leading concept for mRNA delivery. Unsaturated lipids play important roles in nature with potential for mRNA therapeutics, but are difficult to access through chemical synthesis. To systematically study the role of unsaturation, modular reactions were utilized to access a library of 91 amino lipids, enabled by the synthesis of unsaturated thiols. An ionizable lipid series (4A3) emerged from in vitro and in vivo screening, where the 4A3 core with a citronellol-based (Cit) periphery emerged as best. We studied the interaction between LNPs and a model endosomal membrane where 4A3-Cit demonstrated superior lipid fusion over saturated lipids, suggesting its unsaturated tail promotes endosomal escape. Furthermore, 4A3-Cit significantly improved mRNA delivery efficacy in vivo through Selective ORgan Targeting (SORT), resulting in 18-fold increased protein expression over parent LNPs. These findings provide insight into how lipid unsaturation promotes mRNA delivery and demonstrate how lipid mixing can enhance efficacy.