RESUMEN
Early endosomes are the sorting hub on the endocytic pathway, wherein sorting nexins (SNXs) play important roles for formation of the distinct membranous microdomains with different sorting functions. Tubular endosomes mediate the recycling of clathrin-independent endocytic (CIE) cargoes back toward the plasma membrane. However, the molecular mechanism underlying the tubule formation is still poorly understood. Here we screened the effect on the ARF-6-associated CIE recycling endosomal tubules for all the SNX members in Caenorhabditis elegans (C. elegans). We identified SNX-3 as an essential factor for generation of the recycling tubules. The loss of SNX-3 abolishes the interconnected tubules in the intestine of C. elegans. Consequently, the surface and total protein levels of the recycling CIE protein hTAC are strongly decreased. Unexpectedly, depletion of the retromer components VPS-26/-29/-35 has no similar effect, implying that the retromer trimer is dispensable in this process. We determined that hTAC is captured by the ESCRT complex and transported into the lysosome for rapid degradation in snx-3 mutants. Interestingly, EEA-1 is increasingly recruited on early endosomes and localized to the hTAC-containing structures in snx-3 mutant intestines. We also showed that SNX3 and EEA1 compete with each other for binding to phosphatidylinositol-3-phosphate enriching early endosomes in Hela cells. Our data demonstrate for the first time that PX domain-only C. elegans SNX-3 organizes the tubular endosomes for efficient recycling and retrieves the CIE cargo away from the maturing sorting endosomes by competing with EEA-1 for binding to the early endosomes. However, our results call into question how SNX-3 couples the cargo capture and membrane remodeling in the absence of the retromer trimer complex.
Asunto(s)
Factores de Ribosilacion-ADP/genética , Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Endosomas/metabolismo , Células Epiteliales/metabolismo , Nexinas de Clasificación/genética , Proteínas de Transporte Vesicular/genética , Factor 6 de Ribosilación del ADP , Factores de Ribosilacion-ADP/metabolismo , Animales , Transporte Biológico , Caenorhabditis elegans/citología , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Membrana Celular/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Células Epiteliales/citología , Regulación de la Expresión Génica , Células HeLa , Humanos , Intestinos/citología , Lisosomas/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteolisis , Transducción de Señal , Nexinas de Clasificación/deficiencia , Proteínas de Transporte Vesicular/metabolismoRESUMEN
DNA helix-based HIV-1 fusion inhibitors have been discovered as potent drug candidates. Introduction of hydrophobic groups to a nucleobase provides an opportunity to design inhibitors with novel structures and mechanisms of action. In this work, two novel nucleoside analogues (1 and 2) were synthesized and incorporated into four DNA duplex- and quadruplex-based inhibitors. All the molecules showed anti-HIV-1 fusion activity. The effect of the p-benzyloxyphenyl group and the attached linker on the helix formation and thermal stability were fully compared and discussed. Surface plasmon resonance analysis further indicated that inhibitors with the same DNA helix may still have variable reaction targets, mainly attributed to the different hydrophobic modifications.
Asunto(s)
ADN/metabolismo , Inhibidores de Fusión de VIH/síntesis química , Nucleósidos/química , Dicroismo Circular , ADN/química , Proteína gp41 de Envoltorio del VIH/antagonistas & inhibidores , Proteína gp41 de Envoltorio del VIH/metabolismo , Inhibidores de Fusión de VIH/metabolismo , Inhibidores de Fusión de VIH/farmacología , VIH-1/efectos de los fármacos , Humanos , Cinética , Resonancia por Plasmón de Superficie , Temperatura de TransiciónRESUMEN
Short peptide-based inhibition of fusion remains an attractive goal in antihuman immunodeficiency virus (HIV) research based on its potential for the development of technically and economically desirable antiviral agents. Herein, we report the use of the dithiol bisalkylation reaction to generate a series of m-xylene thioether-stapled 22-residue α-helical peptides that have been identified as fusion inhibitors targeting HIV-1 glycoprotein 41 (gp41). The peptide sequence is based on the helix-zone binding domain of the gp41 C-terminal heptad repeat region. We found that one of these stapled peptides, named hCS6ERE, showed promising inhibitory potency against HIV-1 Env-mediated cell-cell fusion and viral replication at a level comparable to the clinically used 36-mer peptide T20. Furthermore, combining hCS6ERE with a fusion inhibitor having a different target site, such as HP23, produced synergistic anti-HIV-1 activity. Collectively, our study offers new insight into the design of anti-HIV peptides with short sequences.
Asunto(s)
Diseño de Fármacos , Proteína gp41 de Envoltorio del VIH/química , Inhibidores de Fusión de VIH/química , Péptidos/química , Sulfuros/química , Secuencia de Aminoácidos , Animales , Endopeptidasa K/metabolismo , Proteína gp41 de Envoltorio del VIH/metabolismo , Inhibidores de Fusión de VIH/metabolismo , Inhibidores de Fusión de VIH/farmacología , VIH-1/fisiología , Humanos , Hígado/metabolismo , Péptidos/metabolismo , Péptidos/farmacología , Unión Proteica , Conformación Proteica en Hélice alfa , Ratas , Internalización del Virus/efectos de los fármacos , Replicación Viral/efectos de los fármacos , Xilenos/químicaRESUMEN
DNA helix-based HIV-1 fusion inhibitors have been discovered as potent drug candidates, but further research is required to enhance their efficiency. The trimeric structure of the HIV-1 envelope glycoprotein provides a structural basis for multivalent drug design. In this work, a "multi-domain" strategy was adopted for design of an oligodeoxynucleotide with assembly, linkage, and activity domains. Built on the self-assembly of higher-order nucleic acid structure, a novel category of multivalent DNA helix-based HIV-1 fusion inhibitor could be easily obtained by a simple annealing course in solution buffer, with no other chemical synthesis for multivalent connection. An optimized multivalent molecule, M4, showed significantly higher anti-HIV-1 fusion activity than did corresponding monovalent inhibitors. Examination of the underlying mechanism indicated that M4 could interact with HIV-1 glycoproteins gp120 and gp41, thereby inhibiting 6HB formation in the fusion course. M4 also showed anti-RDDP and anti-RNase H activity of reverse transcriptase. Besides, these assembled molecules showed improved in vitro metabolic stability in liver homogenate, kidney homogenate, and rat plasma. Moreover, little acute toxicity was observed. Our findings aid in the structural design and understanding of the mechanisms of DNA helix-based HIV-1 inhibitors. This study also provides a general strategy based on a new structural paradigm for the design of other multivalent nucleic acid drugs.
Asunto(s)
Inhibidores de Fusión de VIH/administración & dosificación , VIH-1/efectos de los fármacos , Oligodesoxirribonucleótidos/administración & dosificación , Animales , Proteína gp120 de Envoltorio del VIH/metabolismo , Proteína gp41 de Envoltorio del VIH/metabolismo , Inhibidores de Fusión de VIH/farmacocinética , Inhibidores de Fusión de VIH/toxicidad , VIH-1/fisiología , Riñón/metabolismo , Hígado/metabolismo , Masculino , Ratones Endogámicos ICR , Oligodesoxirribonucleótidos/farmacocinética , Oligodesoxirribonucleótidos/toxicidad , Ratas Sprague-Dawley , Internalización del Virus/efectos de los fármacosRESUMEN
The hexameric α-helical coiled-coil formed between the C-terminal and N-terminal heptad repeat (CHR and NHR) regions of class I viral fusion proteins plays an important role in mediating the fusion of the viral and cellular membranes and provides a clear starting point for molecular mimicry that drives viral fusion inhibitor design. Unfortunately, such peptide mimicry of the short α-helical region in the CHR of Middle East respiratory syndrome coronavirus (MERS-CoV) spike protein has been thwarted by the loss of the peptide's native α-helical conformation when taken out of the parent protein structure. Here, we describe that appropriate all-hydrocarbon stapling of the short helical portion-based peptide to reinforce its bioactive secondary structure remarkably improves antiviral potency. The resultant stapled peptide P21S10 could effectively inhibit infection by MERS-CoV pseudovirus and its spike protein-mediated cell-cell fusion; additionally, P21S10 exhibits improved pharmacokinetic properties than HR2P-M2, suggesting strong potential for development as an anti-MERS-CoV therapeutic.
Asunto(s)
Antivirales/química , Infecciones por Coronavirus/prevención & control , Coronavirus del Síndrome Respiratorio de Oriente Medio/efectos de los fármacos , Péptidos/farmacología , Antivirales/farmacología , Descubrimiento de Drogas , Humanos , Hidrocarburos/química , Péptidos/química , Péptidos/farmacocinética , Conformación Proteica en Hélice alfa , Internalización del Virus/efectos de los fármacosRESUMEN
ETHNOPHARMACOLOGICAL RELEVANCE: Uncaria rhynchophylla (Miq.) Miq. ex Havil., is a plant species used in traditional Chinese medicine to treat cardiovascular and central nervous system diseases. Rhynchophylline (RIN) and isorhynchophylline (IRN), a pair of epimers, are major alkaloids isolated from U. rhynchophylla and exhibit diverse pharmacological effects. Our previous study demonstrated that the pharmacokinetics of these epimers existed stereoselectivity after oral administration; however, the specific mechanism remains unknown and merits investigation. AIM OF THE STUDY: In the present study, the aim was to elucidate the mechanism underlying stereoselective pharmacokinetic characteristics of RIN and IRN in rats. MATERIALS AND METHODS: The total (F), hepatic (Fh) and intestinal (Fa·Fg) bioavailabilities of each epimer were measured using portal vein cannulated rats following different dosing routes (intravenous, intraportal and intraduodenal) to assess individual contributions of the liver and intestine in stereoselective pharmacokinetics. Then the differences of first-pass metabolism in the liver and intestine between two epimers were evaluated by in vitro incubation with rat liver microsomes, intestinal S9 and gastrointestinal (GI) content solutions, respectively. Meanwhile, the membrane permeability and efflux by P-glycoprotein (P-gp) were examined by in situ single-pass intestinal perfusion with and without P-gp inhibitor verapamil. The configurational interconversion at different pH values and the excretions via feces and urine were also examined. RESULTS: Pharmacokinetic data showed that the total bioavailability of RIN was 5.9 folds higher than that of IRN (23.4% vs. 4.0%). The hepatic availability of RIN was 4.6 folds higher than that of IRN (46.9% vs. 10.3%), whereas the intestinal availability of RIN (48.1%) was comparable to that of IRN (42.7%). In addition, intestinal perfusion showed that IRN possessed higher intestinal permeability than RIN and co-perfusion with verapamil could affect absorption process of RIN but not IRN. Conversely, the metabolism rate of IRN in rat liver microsomes was significantly faster than that of RIN, resulting in a lower systemic exposure of IRN after oral administration. The degradation in GI lumen and epimerization between two epimers also existed but had small contributions. Additionally, the excretions of both epimers via feces and urine were negligible. CONCLUSIONS: Taken together, different first-pass metabolism in the liver was the major factor responsible for the stereoselective pharmacokinetics of RIN and IRN.
Asunto(s)
Alcaloides Indólicos/farmacocinética , Mucosa Intestinal/metabolismo , Hígado/metabolismo , Animales , Área Bajo la Curva , Semivida , Alcaloides Indólicos/sangre , Alcaloides Indólicos/química , Alcaloides Indólicos/metabolismo , Masculino , Microsomas Hepáticos/metabolismo , Estructura Molecular , Oxindoles , Ratas , Ratas Sprague-DawleyRESUMEN
Endocytosis and postendocytic sorting of G-protein-coupled receptors (GPCRs) is important for the regulation of both their cell surface density and signaling profile. Unlike the mechanisms of clathrin-dependent endocytosis (CDE), the mechanisms underlying the control of GPCR signaling by clathrin-independent endocytosis (CIE) remain largely unknown. Among the muscarinic acetylcholine receptors (mAChRs), the M4 mAChR undergoes CDE and recycling, whereas the M2 mAChR is internalized through CIE and targeted to lysosomes. Here we investigated the endocytosis and postendocytic trafficking of M2 mAChR based on a comparative analysis of the third cytoplasmic domain in M2 and M4 mAChRs. For the first time, we identified that the sequence (374)KKKPPPS(380) servers as a sorting signal for the clathrin-independent internalization of M2 mAChR. Switching (374)KKKPPPS(380) to the i3 loop of the M4 mAChR shifted the receptor into lysosomes through the CIE pathway; and therefore away from CDE and recycling. We also found another previously unidentified sequence that guides CDE of the M2 mAChR, (361)VARKIVKMTKQPA(373), which is normally masked in the presence of the downstream sequence (374)KKKPPPS(380). Taken together, our data indicate that endocytosis and postendocytic sorting of GPCRs that undergo CIE could be sequence-dependent.