RESUMEN
A sodium alginate (Alg) based REDOX (reduction and oxidation)-responsive and fluorescent active microgel was prepared via water in oil (w/o) mini-emulsion polymerization technique. Here, we initially synthesized sodium alginate-based disulfide cross linked microgels and after that those microgels were tagged with rhodamine amine derivative (RhB-NH2) by ionic interaction to get the pH-responsive fluorescent property. Functionalized microgels were characterized using 1H NMR, FTIR, DLS, HRTEM, FESEM, UV-vis, and fluorescence spectroscopy analyses. Presence of the REDOX-responsive disulfide-containing crosslinkers in the microgels enhances the release of doxorubicin (DOX), an anti-cancer drug in the reducing environment of the cancer-cells (simulated). Existence of the rhodamine-amine derivative in the microgels triggers the pH-dependent fluorescence property by showing fluorescence emission at 560-580 nm at pH 5.5 (cancer cell pH). The cytotoxicity of the biopolymer based microgel was assessed over both cancerous HeLa (IC50 100 µg/mL) and non-cancerous MDCK (IC50 200 µg/mL) cells by MTT assay which showed the synthesized microgel is non-toxic whereas DOX-loaded microgels showed significant toxicity. FACS and cell uptake (in vitro) analyses were conducted to understand the cell apoptosis cycle and behavior of the cancer cells in presence of the DOX-loaded microgels. This pH-responsive fluorescent active alginate-based biomaterial could be a promising material for the anti-cancer drug delivery and other medical fields.
Asunto(s)
Alginatos , Doxorrubicina , Sistemas de Liberación de Medicamentos , Liberación de Fármacos , Microgeles , Oxidación-Reducción , Alginatos/química , Concentración de Iones de Hidrógeno , Doxorrubicina/administración & dosificación , Doxorrubicina/farmacología , Doxorrubicina/química , Humanos , Células HeLa , Animales , Microgeles/química , Sistemas de Liberación de Medicamentos/métodos , Perros , Antineoplásicos/química , Antineoplásicos/administración & dosificación , Antineoplásicos/farmacología , Rodaminas/química , Supervivencia Celular/efectos de los fármacos , Portadores de Fármacos/química , Colorantes Fluorescentes/químicaRESUMEN
Influenza viruses remain a major public health concern causing contagious respiratory illnesses that result in around 290,000-650,000 global deaths every year. Their ability to constantly evolve through antigenic shifts and drifts leads to the emergence of newer strains and resistance to existing drugs and vaccines. To combat this, there is a critical need for novel antiviral drugs through the introduction of host-targeted therapeutics. Influenza viruses encode only 14 gene products that get extensively modified through phosphorylation by a diverse array of host kinases. Reversible phosphorylation at serine, threonine, or tyrosine residues dynamically regulates the structure, function, and subcellular localization of viral proteins at different stages of their life cycle. In addition, kinases influence a plethora of signaling pathways that also regulate virus propagation by modulating the host cell environment thus establishing a critical virus-host relationship that is indispensable for executing successful infection. This dependence on host kinases opens up exciting possibilities for developing kinase inhibitors as next-generation anti-influenza therapy. To fully capitalize on this potential, extensive mapping of the influenza virus-host kinase interaction network is essential. The key focus of this review is to outline the molecular mechanisms by which host kinases regulate different steps of the influenza A virus life cycle, starting from attachment-entry to assembly-budding. By assessing the contributions of different host kinases and their specific phosphorylation events during the virus life cycle, we aim to develop a holistic overview of the virus-host kinase interaction network that may shed light on potential targets for novel antiviral interventions.
Asunto(s)
Interacciones Huésped-Patógeno , Gripe Humana , Proteínas Quinasas , Transducción de Señal , Humanos , Virus de la Influenza A/genética , Virus de la Influenza A/fisiología , Gripe Humana/metabolismo , Replicación Viral , Proteínas Quinasas/metabolismo , FosforilaciónRESUMEN
We report a nucleic acid-based point of care testing technology for infectious disease detection at resource limited settings by integrating a low-cost portable device with machine learning-empowered quantitative colorimetric analytics that can be interfaced via a smartphone application. We substantiate our proposition by demonstrating the efficacy of this technology in detecting COVID-19 infection from human swab samples, using the RT-LAMP protocol. Comparison with gold standard results from real-time PCR evidences high sensitivity and specificity, ensuring simplicity, portability, and user-friendliness of the technology at the same time. Colorimetric analytics of the reaction output without necessitating the opening of the reaction microchambers enables execution of the complete test workflow without any laboratory control that may otherwise be required stringently for safeguarding against carryover contamination. Seamless sample-to-answer workflow and machine learning-based readout further assures minimal human intervention for the test readout, thus eliminating inevitable inaccuracies stemming from erroneous execution of the test as well as subjectivity in interpreting the outcome. Our results further indicate the possibilities of upgrading the technology to predict the pathogenic load on the infected patients akin to the cyclic threshold value of the real-time PCR, when calibrated with reference to a wide range of 'training' data for the machine learner, thereby putting forward the same as viable alternative to the resource-intensive PCR tests that cannot be made readily accessible at underserved community settings.
Asunto(s)
Enfermedades Transmisibles , Ácidos Nucleicos , Humanos , Colorimetría , Teléfono Inteligente , Pruebas en el Punto de Atención , TecnologíaRESUMEN
Frequent mutation and variable immunological protection against vaccination is a common feature for COVID-19 pandemic. Early detection and confinement remain key to controlling further spread of infection. In response, we have developed an aptamer-based system that possesses both diagnostic and therapeutic potential towards the virus. A random aptamer library (~ 1017 molecules) was screened using systematic evolution of ligands by exponential enrichment (SELEX) and aptamer R was identified as a potent binder for the SARS-CoV-2 spike receptor binding domain (RBD) using in vitro binding assay. Using a pseudotyped viral entry assay we have shown that aptamer R specifically inhibited the entry of a SARS-CoV-2 pseudotyped virus in HEK293T-ACE2 cells but did not inhibit the entry of a Vesicular Stomatitis Virus (VSV) glycoprotein (G) pseudotyped virus, hence establishing its specificity towards SARS-CoV-2 spike protein. The antiviral potential of aptamers R and J (same central sequence as R but lacking flanked primer regions) was tested and showed 95.4% and 82.5% inhibition, respectively, against the SARS-CoV-2 virus. Finally, intermolecular interactions between the aptamers and the RBD domain were analyzed using in silico docking and molecular dynamics simulations that provided additional insight into the binding and inhibitory action of aptamers R and J.
Asunto(s)
COVID-19 , Inhibidores de Fusión de VIH , Humanos , SARS-CoV-2 , Células HEK293 , Pandemias , Ligandos , Oligonucleótidos , Prueba de COVID-19RESUMEN
The efficient monitoring and early detection of viruses may provide essential information about diseases. In this work, we have highlighted the interaction between DNA and a two-dimensional (2D) metal oxide for developing biosensors for further detection of viral infections. Spectroscopic measurements have been used to probe the efficient interactions between single-stranded DNA (ssDNA) and the 2D metal oxide and make them ideal candidates for detecting viral infections. We have also used fully atomistic molecular dynamics (MD) simulation to give a microscopic understanding of the experimentally observed ssDNA-metal oxide interaction. The adsorption of ssDNA on the inorganic surface was found to be driven by favourable enthalpy change, and 5'-guanine was identified as the interacting nucleotide base. Additionally, the in silico assessment of the conformational changes of the ssDNA chain during the adsorption process was also performed in a quantitative manner. Finally, we comment on the practical implications of these developments for sensing that could help design advanced systems for preventing virus-related pandemics.
Asunto(s)
Técnicas Biosensibles , Virus , ADN , ADN de Cadena Simple , Técnicas Biosensibles/métodos , Óxidos/química , Simulación de Dinámica MolecularRESUMEN
Background: Hyper-inflammatory immune response of SARS-CoV-2 is often characterized by the release of multiple pro-inflammatory cytokines with an impact on the expression of numerous other interleukins (ILs). However, from oral and nasal swab samples the specific quantitative association of the different IL-markers with the disease progression and its relationship with the status of vaccination remains unclear. Materials and methods: Patients' combined oral and nasal swab samples were collected from both non-vaccinated and double-vaccinated individuals with high (Ct value < 25) and low (Ct value > 30) viral loads, along with uninfected donors. None of the patients were critically ill, or needed ICU support. The expression of different cytokines (IL6, IL10, IL1B, IFNG) and mucin (MUC5AC, MUC1) markers were assessed between different groups by qRT-PCR. The important cytokine markers differentiating between vaccinated and non-vaccinated patients were identified by PCA. Conclusion: IL6 expression was higher in non-vaccinated COVID-19 patients infected with delta-variant irrespective of their viral-load compared to uninfected individuals. However, in double-vaccinated patients, only in high viral-load patients (Ct value < 25), IL6 expression increased. In high viral-load patients, irrespective to their vaccination status, IL10 expression was lower compared to the uninfected control group. Surprisingly, IL10 expression was lower in double-vaccinated patients with Ct value > 30. IL1B, and IFNG expression remained unaltered in uninfected and infected individuals. However, MUC5AC expression was lower in non-vaccinated patients with Ct value < 25 compared to control group. Our study unveiled that IL10/IL6 ratio can be used as a biomarker for COVID-19 patients upon proper establishment of it in a clinical setting.
RESUMEN
Throughout a few years, carrier transport studies across HaP single crystals have gained enormous importance for current generation photovoltaic and photodetector research with their superior optoelectronic properties compared to commercially available polycrystalline materials. Utilizing the room-temperature solution-grown method, we synthesized MAPbBr3 crystals and examined their electrical transport properties. Although the X-ray diffraction reveals the cubical nature of the crystals, we have observed anisotropy in the electrical transport behavior and variation in dielectric constant across the three opposite faces of the crystals of mm dimensions. The face with a higher dielectric constant depicts improved parameters from electrical characteristics such as lower trap densities and higher mobility values. We further explore the origin of its anisotropic nature by performing X-ray diffraction on three opposite faces of crystals. Our studies define the specific faces of cuboid-shaped MAPbBr3 crystals for efficient electrical contact in the fabrication of optoelectronic devices.
RESUMEN
The imidazolium compound Ym155 was first reported to be a survivin inhibitor. Ym155 potently induces cell death of many types of cancer cells in preclinical studies. However, in phase II clinical trials Ym155 failed to demonstrate a significant benefit. Studies have suggested that the cytotoxic effects of Ym155 in cancer cells are not mediated by the inhibition of survivin. Understanding the mechanism by which Ym155 induces cell death would provide important insight how to improve its efficacy as a cancer therapeutic. We demonstrate a novel mechanism by which Ym155 induces cell death by localizing to the mitochondria causing mitochondrial dysfunction. Our studies suggest that Ym155 binds mitochondrial DNA leading to a decrease in oxidative phosphorylation, decrease in TCA cycle intermediates, and an increase in mitochondrial permeability. Furthermore, we show that mitochondrial stress induced by Ym155 and other mitochondrial inhibitors activates AMP-activated kinase leading to the downregulation to bone morphogenetic protein (BMP) signaling. We provide first evidence that Ym155 initiates cell death by disrupting mitochondrial function.
Asunto(s)
Antineoplásicos , Imidazoles/farmacología , Neoplasias Pulmonares , Naftoquinonas/farmacología , Proteínas Quinasas Activadas por AMP , Antineoplásicos/farmacología , Apoptosis , Proteínas Morfogenéticas Óseas/metabolismo , Línea Celular Tumoral , Humanos , Proteínas Inhibidoras de la Apoptosis/metabolismo , Neoplasias Pulmonares/tratamiento farmacológico , Mitocondrias/metabolismo , Survivin/metabolismoRESUMEN
BACKGROUND: Bone morphogenetic protein (BMP) is a phylogenetically conserved signaling pathway required for development that is aberrantly expressed in several age-related diseases including cancer, Alzheimer's disease, obesity, and cardiovascular disease. Aberrant BMP signaling in mice leads to obesity, suggesting it may alter normal metabolism. The role of BMP signaling regulating cancer metabolism is not known. METHODS: To examine BMP regulation of metabolism, C. elegans harboring BMP gain-of-function (gof) and loss-of-function (lof) mutations were examined for changes in activity of catabolic and anabolic metabolism utilizing Western blot analysis and fluorescent reporters. AMP activated kinase (AMPK) gof and lof mutants were used to examine AMPK regulation of BMP signaling. H1299 (LKB1 wild-type), A549 (LKB1 lof), and A549-LKB1 (LKB1 restored) lung cancer cell lines were used to study BMP regulation of catabolic and anabolic metabolism. Studies were done using recombinant BMP ligands to activate BMP signaling, and BMP receptor specific inhibitors and siRNA to inhibit signaling. RESULTS: BMP signaling in both C. elegans and cancer cells is responsive to nutrient conditions. In both C. elegans and lung cancer cell lines BMP suppressed AMPK, the master regulator of catabolism, while activating PI3K, a regulator of anabolism. In lung cancer cells, inhibition of BMP signaling by siRNA or small molecules increased AMPK activity, and this increase was mediated by activation of LKB1. BMP2 ligand suppressed AMPK activation during starvation. BMP2 ligand decreased expression of TCA cycle intermediates and non-essential amino acids in H1299 cells. Furthermore, we show that BMP activation of PI3K is mediated through BMP type II receptor. We also observed feedback signaling, as AMPK suppressed BMP signaling, whereas PI3K increased BMP signaling. CONCLUSION: These studies show that BMP signaling suppresses catabolic metabolism and stimulates anabolic metabolism. We identified feedback mechanisms where catabolic induced signaling mediated by AMPK negatively regulates BMP signaling, whereas anabolic signaling produces a positive feedback regulation of BMP signing through Akt. These mechanisms were conserved in both lung cancer cells and C. elegans. These studies suggest that aberrant BMP signaling causes dysregulation of metabolism that is a potential mechanism by which BMP promotes survival of cancer cells.
RESUMEN
The mini-genome reporter assay is a key tool for conducting RNA virus research. However, procedural complications and the lack of adequate literature pose a major challenge in developing these assay systems. Here, we present a novel, yet generic and simple, cloning strategy for the construction of an influenza B virus reporter RNA template and describe an extensive standardization of the reporter RNP/polymerase activity assay for monitoring viral RNA synthesis in an infection-free setting. Using this assay system, we showed for the first time the effect of viral protein NS1 and host protein kinase C delta (PKCD) on influenza B virus RNA synthesis. In addition, the assay system showed promising results in evaluating the efficacy of antiviral drugs targeting viral RNA synthesis and virus propagation. Together, this work offers a detailed protocol for the standardization of the influenza virus minigenome assay and an excellent tool for screening of host factors and antivirals in a fast, user-friendly, and high-throughput manner.
RESUMEN
BACKGROUND: Bone morphogenetic proteins (BMP) are evolutionarily conserved morphogens that are reactivated in lung carcinomas. In lung cancer cells, BMP signaling suppresses AMP activated kinase (AMPK) by inhibiting LKB1. AMPK is activated by mitochondrial stress that inhibits ATP production, which is enhanced 100-fold when phosphorylated by LKB1. Activated AMPK can promote survival of cancer cells but its "hyperactivation" induces cell death. The studies here reveal novel cell death mechanisms induced by BMP inhibitors, together with agents targeting the mitochondria, which involves the "hyperactivation" of AMPK. METHODS: This study examines the synergistic effects of two BMP inhibitors together with mitochondrial targeting agents phenformin and Ym155, on cell death of lung cancer cells expressing LKB1 (H1299), LKB1 null (A549), and A549 cells transfected with LKB1 (A549-LKB1). Cell death mechanisms evaluated were the activation of caspases and the nuclear localization of apoptosis inducing factor (AIF). A769662 was used to allosterically activate AMPK. Knockdown of BMPR2 and LKB1 using siRNA was used to examine their effects on nuclear localization of AMPK. Validation studies were performed on five passage zero primary NSCLC. RESULTS: Both BMP inhibitors synergistically suppressed growth when combined with Ym155 or phenformin in cells expressing LKB1. The combination of BMP inhibitors with mitochondrial targeting agents enhanced the activation of AMPK in lung cancer cells expressing LKB1. Allosteric activation of AMPK with A769662 induced cell death in both H1299 and A549 cells. Cell death induced by the combination of BMP inhibitors and mitochondrial-targeting agents did not activate caspases. The combination of drugs induced nuclear localization of AIF in cells expressing LKB1, which was attenuated by knockdown of LKB1. Knockdown of BMPR2 together with Ym155 increased nuclear localization of AIF. Combination therapy also enhanced cell death and AIF nuclear localization in primary NSCLC. CONCLUSIONS: These studies demonstrate that inhibition of BMP signaling together with mitochondrial targeting agents induce AIF caspase-independent cell death, which involves the "hyperactivation" of AMPK. AIF caspase-independent cell death is an evolutionarily conserved cell death pathway that is infrequently studied in cancer. These studies provide novel insight into mechanisms inducing AIF caspase-independent cell death in cancer cells using BMP inhibitors. Video Abstract.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Proteínas Quinasas Activadas por AMP/metabolismo , Apoptosis , Factor Inductor de la Apoptosis/metabolismo , Proteínas Morfogenéticas Óseas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Caspasas/metabolismo , Caspasas/farmacología , Muerte Celular , Humanos , Pulmón/metabolismo , Neoplasias Pulmonares/patología , Mitocondrias/metabolismo , Fenformina/metabolismo , Fenformina/farmacología , Proteínas Serina-Treonina QuinasasRESUMEN
As newer variants of SARS-CoV-2 continue to pose major threats to global human health and economy, identifying novel druggable antiviral targets is the key towards sustenance. Here, we identify an evolutionary conserved E-L-L motif present within the HR2 domain of all human and non-human coronavirus spike (S) proteins that play a crucial role in stabilizing the post-fusion six-helix bundle (6-HB) structure and thus, fusion-mediated viral entry. Mutations within this motif reduce the fusogenicity of the S protein without affecting its stability or membrane localization. We found that posaconazole, an FDA-approved drug, binds to this E-L-L motif resulting in effective inhibition of SARS-CoV-2 infection in cells. While posaconazole exhibits high efficacy towards blocking S protein-mediated viral entry, mutations within the E-L-L motif rendered the protein completely resistant to the drug, establishing its specificity towards this motif. Our data demonstrate that posaconazole restricts early stages of infection through specific inhibition of membrane fusion and viral genome release into the host cell and is equally effective towards all major variants of concerns of SARS-CoV-2 including beta, kappa, delta, and omicron. Together, we show that this conserved essential E-L-L motif is an ideal target for the development of prophylactic and therapeutic interventions against SARS-CoV-2.
RESUMEN
Developing green or red light-activated drug delivery systems (DDSs) for cancer treatment is highly desirable. Herein, we have reported a green light-responsive single component-based organic fluorescence nano-DDS by simply anchoring 2-hydroxy-6-naphthacyl (phototrigger) on both sides of the 1,5-diaminonaphthalene (DAN) chromophore. This green light (λ ≥ 500 nm)-activated DDS released two equivalents of the anticancer drug (valproic acid) in a spatio-temporally controlled manner. Our photoresponsive DDS [DAN-bis(HO-Naph-VPA)] exhibited interesting properties such as excited-state intramolecular proton transfer (ESIPT) accompanied with aggregation-induced emission (AIE) phenomena. AIE initiated the photorelease, and ESIPT enhanced the rate of the photorelease. Further, in vitro studies revealed that our green light-activated nano-DDS exhibited good cytocompatibility, excellent cellular internalization, and effective cancer cell killing ability.
Asunto(s)
Antineoplásicos , Sistema de Administración de Fármacos con Nanopartículas , Antineoplásicos/farmacología , Sistemas de Liberación de Medicamentos , Fluorescencia , ProtonesRESUMEN
Glioblastomas (GBMs) are aggressive brain tumors that are resistant to chemotherapy and radiation. Bone morphogenetic protein (BMP) ligand BMP4 is being examined as a potential therapeutic for GBMs because it induces differentiation of cancer stem cells (CSCs) to an astrocyte phenotype. ID1 is reported to promote self-renewal and inhibit CSC differentiation. In most cancers, ID1 is transcriptionally upregulated by BMP4 promoting invasion and stemness. This conflicting data bring into question whether BMP signaling is growth suppressive or growth promoting in GBMs. We utilized BMP inhibitors DMH1, JL5, and Ym155 to examine the role of BMP signaling on the growth of GBMs. DMH1 targets BMP type 1 receptors whereas JL5 inhibits both the type 1 and type 2 BMP receptors. Ym155 does not bind the BMP receptors but rather inhibits BMP signaling by inducing the degradation of BMPR2. We show that JL5, DMH1, and Ym155 decreased the expression of ID1 in SD2 and U87 cells. JL5 and Ym155 also decreased the expression of BMPR2 and its downstream target inhibitor of apoptosis protein XIAP. JL5 treatment resulted in significant cell death and suppressed self-renewal to a greater extent than that induced by BMP4 ligand. The lysosome inhibitor chloroquine increases the localization of BMPR2 to the plasma membrane enhancing JL5-induced downregulation of ID1 and cell death in SD2 cells. We show that BMP signaling is growth promoting in GBMs. These studies suggest the need for development of BMP inhibitors and evaluation as potential therapeutic for GBMs.
Asunto(s)
Glioblastoma , Proteína Morfogenética Ósea 4 , Receptores de Proteínas Morfogenéticas Óseas , Proteínas Morfogenéticas Óseas , Glioblastoma/tratamiento farmacológico , Humanos , Ligandos , Transducción de SeñalRESUMEN
Bat influenza viruses are genetically distant from classical influenza A viruses (IAVs) and show distinct functional differences in their surface antigens. Nevertheless, any comparative analyses between bat and classical IAV RNA polymerases or their specific subunits are yet to be performed. In this work, we have identified signature residues present in the bat influenza virus polymerase which are responsible for its altered fitness in comparison to the classical IAVs. Through comparative sequence and structural analysis, we have identified specific positions in the PB2 subunit of the polymerase, with differential amino acid preferences among bat and nonbat IAVs. Functional screening helped us to focus upon the previously uncharacterized PB2-282 residue, which is serine in bat virus but harbors highly conserved glutamic acid in classical IAVs. Introduction of E282S mutation in the human-adapted PB2 (influenza A/H1N1/WSN/1933) drastically reduces polymerase activity and replication efficiency of the virus in human, bat, and canine cells. Interestingly, this newly identified PB2-282 residue within an evolutionary conserved "S-E-S" motif, present across different genera of influenza viruses and serving as a key regulator of RNA synthesis activity of the polymerase. In contrast, bat influenza viruses harbor an atypical "S-S-T" motif at the same position of PB2, alteration of which with the human-like "S-E-T" motif significantly enhances its (H17N10/Guatemala/164/2009) polymerase activity in human cells. Together, our data indicate that the PB2-S282 residue may serve as an inherent restriction element of the bat virus polymerase, limiting its activity in other host species. IMPORTANCE Influenza A viruses are known for their ability to perform cross-species transmission, facilitated by amino acid alterations either in the surface antigen hemagglutinin (HA) or in the polymerase subunit PB2. Recent isolation of influenza A-like viruses from bats raised concern about their epizootic and zoonotic potential. Here, we identify a novel species-specific signature present within the influenza virus polymerase that may serve as a key factor in adaptation of influenza viruses from bat to nonbat host species. The PB2-282 residue, which harbors a highly conserved glutamic acid for influenza viruses across all genera (A, B, C, and D), encompasses an atypical serine in the case of bat influenza viruses. Our data show that the human-adapted polymerase, harboring a bat-specific signature (PB2-S282,) performs poorly, while bat PB2 protein, harboring a human-specific signature (PB2-E282), shows increased fitness in human cells.
Asunto(s)
Virus de la Influenza A , Infecciones por Orthomyxoviridae , ARN Polimerasa Dependiente del ARN , Proteínas Virales , Adaptación Fisiológica/genética , Secuencias de Aminoácidos , Animales , Línea Celular , Quirópteros , Perros , Humanos , Virus de la Influenza A/genética , Virus de la Influenza A/metabolismo , Infecciones por Orthomyxoviridae/transmisión , Infecciones por Orthomyxoviridae/virología , ARN/metabolismo , ARN Polimerasa Dependiente del ARN/química , ARN Polimerasa Dependiente del ARN/genética , ARN Polimerasa Dependiente del ARN/metabolismo , Especificidad de la Especie , Proteínas Virales/química , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
As newer variants of SARS-CoV-2 continue to pose major threats to global human health and economy, identifying novel druggable antiviral targets is the key toward sustenance. Here, we identify an evolutionarily conserved "Ex3Lx6L" ("E-L-L") motif present within the HR2 domain of all human and nonhuman coronavirus spike (S) proteins that play a crucial role in stabilizing its postfusion six-helix bundle (6-HB) structure and thus, fusion-mediated viral entry. Mutations within this motif reduce the fusogenicity of the S protein without affecting its stability or membrane localization. We found that posaconazole, an FDA-approved drug, binds to this "E-L-L" motif and impedes the formation of 6-HB, thus effectively inhibiting SARS-CoV-2 infection in cells. While posaconazole exhibits high efficacy in blocking S protein-mediated viral entry, mutations within the "E-L-L" motif rendered the protein completely resistant to the drug, establishing its specificity toward this motif. Our data demonstrate that posaconazole restricts early stages of infection through specific inhibition of membrane fusion and viral genome release into the host cell and is equally effective toward all major variants of concerns of SARS-CoV-2, including Beta, Kappa, Delta, and Omicron. Together, we show that this conserved essential "E-L-L" motif is an ideal target for the development of prophylactic and therapeutic interventions against SARS-CoV-2.
RESUMEN
BACKGROUND: Recent studies have shown that bone morphogenetic protein receptor 2 (BMPR2) regulates cell survival signaling events in cancer cells independent of the BMP type 1 receptor (BMPR1) or the Smad-1/5 transcription factor. Mutations in BMPR2 trafficking proteins leads to overactive BMP signaling, which leads to neurological diseases caused by BMPR2 stabilization of the microtubules. It is not known whether BMPR2 regulates the microtubules in cancer cells and what effect this has on cell survival. It is also not known whether alterations in BMPR2 trafficking effects activity and response to BMPR2 inhibitors. METHODS: We utilized BMPR2 siRNA and the BMP receptor inhibitors JL5 and Ym155, which decrease BMPR2 signaling and cause its mislocalization to the cytoplasm. Using the JL5 resistant MDA-MD-468 cell line and sensitive lung cancer cell lines, we examined the effects of BMPR2 inhibition on BMPR2 mislocalization to the cytoplasm, microtubule destabilization, lysosome activation and cell survival. RESULTS: We show that the inhibition of BMPR2 destabilizes the microtubules. Destabilization of the microtubules leads to the activation of the lysosomes. Activated lysosomes further decreases BMPR2 signaling by causing it to mislocalizated to the cytoplasm and/or lysosome for degradation. Inhibition of the lysosomes with chloroquine attenuates BMPR2 trafficking to the lysosome and cell death induced by BMPR2 inhibitors. Furthermore, in MDA-MD-468 cells that are resistant to JL5 induced cell death, BMPR2 was predominately located in the cytoplasm. BMPR2 failed to localize to the cytoplasm and/or lysosome following treatment with JL5 and did not destabilize the microtubules or activate the lysosomes. CONCLUSIONS: These studies reveal that the inhibition of BMPR2 destabilizes the microtubules promoting cell death of cancer cells that involves the activation of the lysosomes. Resistance to small molecules targeting BMPR2 may occur if the BMPR2 is localized predominantly to the cytoplasm and/or fails to localize to the lysosome for degradation. Video Abstract.
Asunto(s)
Receptores de Proteínas Morfogenéticas Óseas de Tipo II/genética , Muerte Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Neoplasias Pulmonares/tratamiento farmacológico , Receptores de Proteínas Morfogenéticas Óseas de Tipo 1/genética , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/antagonistas & inhibidores , Muerte Celular/genética , Supervivencia Celular/efectos de los fármacos , Humanos , Imidazoles/farmacología , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Lisosomas/efectos de los fármacos , Lisosomas/genética , Microtúbulos/efectos de los fármacos , Microtúbulos/genética , Naftoquinonas/farmacología , Pirazoles/farmacología , Pirimidinas/farmacología , Quinolonas/farmacología , ARN Interferente Pequeño/farmacología , Transducción de Señal/efectos de los fármacosRESUMEN
We developed a piecewise isothermal nucleic acid test (PINAT) as a platform technology for diagnosing pathogen-associated infections, empowered by an illustrative novel methodology that embeds an exclusive DNA-mediated specific probing reaction with the backbone of an isothermal reverse transcription cum amplification protocol for detecting viral RNA. In a point-of-care format, this test is executable in a unified single-step, single-chamber procedure, leading to seamless sample-to-result integration in an inexpensive, scalable, pre-programmable, and customizable portable device, with mobile-app-integrated interpretation and analytics involving minimal manually operative procedures. The test exhibited a high sensitivity and specificity of detection when assessed using 200 double-blind patient samples for detecting SARS-CoV-2 infection by the Indian Council of Medical Research (ICMR), and subsequently using 170 double-blind patient samples in a point-of-care format outside controlled laboratory settings as performed by unskilled technicians in an organized clinical trial. We also established its efficacy in detecting Influenza A infection by performing the diagnosis at the point of collection with uncompromised detection rigor. The envisaged trade-off between advanced laboratory-based molecular diagnostic procedures and the elegance of common rapid tests renders the method ideal for deployment in resource-limited settings towards catering the needs of the underserved.
Asunto(s)
COVID-19 , Enfermedades Transmisibles , Humanos , Sistemas de Atención de Punto , ARN Viral/genética , SARS-CoV-2RESUMEN
BACKGROUND/AIM: Tyrosine kinase inhibitors (TKIs) are used for the treatment of both wild type and mutant non-small cell lung cancer (NSCLC); however, acquired resistance is a major clinical challenge. Herein, we aimed to investigate the effects of telmisartan (Tel), CFM 4.16 and sorafenib combination in rociletinib resistant NSCLC tumors. MATERIALS AND METHODS: 3D spheroid cultures and western blotting were used for evaluating cytotoxic effects and protein expression. An in vivo rociletinib resistant H1975 xenograft model of NSCLC was developed by subcutaneous injection of rociletinib resistant H1975 cells into nude mice. RESULTS: Tel, CFM 4.16 and sorafenib combination displayed superior anti-cancer effects in 3D spheroid cultures and a rociletinib resistant H1975 xenograft model of NSCLC by decreasing the protein expression of oncogenic and cancer stem cell markers (Nanog, Sox2 and Oct4). CONCLUSION: Tel facilitates effective penetration of CFM 4.16 and sorafenib in rociletinib resistant H1975 models of NSCLC.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Resistencia a Antineoplásicos/efectos de los fármacos , Neoplasias Pulmonares/tratamiento farmacológico , Esferoides Celulares/citología , Compuestos de Espiro/administración & dosificación , Telmisartán/administración & dosificación , Tiadiazoles/administración & dosificación , Acrilamidas/farmacología , Acrilamidas/uso terapéutico , Animales , Biomarcadores de Tumor/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Regulación hacia Abajo , Sinergismo Farmacológico , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Neoplasias Pulmonares/metabolismo , Ratones , Ratones Desnudos , Pirimidinas/farmacología , Pirimidinas/uso terapéutico , Sorafenib/farmacología , Sorafenib/uso terapéutico , Esferoides Celulares/efectos de los fármacos , Compuestos de Espiro/farmacología , Telmisartán/farmacología , Tiadiazoles/farmacología , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Semiconductor nanoparticles are promising materials for light-driven processes such as solar-fuel generation, photocatalytic pollutant remediation, and solar-to-electricity conversion. Effective application of these materials alongside light can assist in reducing the dependence on fossil-fuel driven processes and aid in resolving critical environmental issues. However, severe recombination of the photogenerated charge-carriers is a persistent bottleneck in several semiconductors, particularly those that contain multiple cations. This issue typically manifests in the form of reduced lifetime of the photoexcited electrons-holes leading to a decrease in the quantum efficiency of various light-driven applications. On the other hand, semiconducting oxides or sulfides, coupled with reduced graphene oxide (RGO), have drawn a considerable interest recently, partly because of the RGO enhancing charge separation and transportation through its honeycomb sp2 network structure. High electron mobility, conductivity, surface area, and cost-effectiveness are the hallmark of the RGO. This Mini-Review focuses on (1) examining the approach to the integration of RGO with semiconductors to produce binary nanocomposites; (2) insights into the microstructure interface, which plays a critical role in leveraging charge transport; (3) key examples of RGO composites with oxide and sulfide semiconductors with photocatalysis as application; and (4) strategies that have to be pursued to fully leverage the benefit of RGO in RGO/semiconductors to attain high photocatalytic activity for a sustainable future. This Mini-Review focuses on areas requiring additional exploration to fully understand the interfacial science of RGO and semiconductor, for clarity regarding the interfacial stability between RGO and the semiconductor, electronic coupling at the heterojunction, and morphological properties of the nanocomposites. We believe that this Mini-Review will assist with streamlining new directions toward the fabrication of RGO/semiconductor nanocomposites with higher photocatalytic activity for solar-driven multifunctional applications.