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Proteostasis mechanisms, such as proteotoxic-stress response and autophagy, are increasingly recognized for their roles in influencing various cancer hallmarks such as tumorigenesis, drug resistance, and recurrence. However, the precise mechanisms underlying their coordination remain not fully elucidated. The aim of this study is to investigate the molecular interplay between Hsp70 and autophagy in lung adenocarcinoma cells and elucidate its impact on the outcomes of anticancer therapies in vitro. For this purpose, we utilized the human lung adenocarcinoma A549 cell line and genetically modified it by knockdown of Hsp70 or HSF1, and the H1299 cell line with knockdown or overexpression of Hsp70. In addition, several treatments were employed, including treatment with Hsp70 inhibitors (VER-155008 and JG-98), HSF1 activator ML-346, or autophagy modulators (SAR405 and Rapamycin). Using immunoblotting, we found that Hsp70 negatively regulates autophagy by directly influencing AMPK activation, uncovering a novel regulatory mechanism of autophagy by Hsp70. Genetic or chemical Hsp70 overexpression was associated with the suppression of AMPK and autophagy. Conversely, the inhibition of Hsp70, genetically or chemically, resulted in the upregulation of AMPK-mediated autophagy. We further investigated whether Hsp70 suppression-mediated autophagy exhibits pro-survival- or pro-death-inducing effects via MTT test, colony formation, CellTiter-Glo 3D-Spheroid viability assay, and Annexin/PI apoptosis assay. Our results show that combined inhibition of Hsp70 and autophagy, along with cisplatin treatment, synergistically reduces tumor cell metabolic activity, growth, and viability in 2D and 3D tumor cell models. These cytotoxic effects were exerted by substantially potentiating apoptosis, while activating autophagy via rapamycin slightly rescued tumor cells from apoptosis. Therefore, our findings demonstrate that the combined inhibition of Hsp70 and autophagy represents a novel and promising therapeutic approach that may disrupt the capacity of refractory tumor cells to withstand conventional therapies in NSCLC.
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Proteínas Quinasas Activadas por AMP , Autofagia , Carcinoma de Pulmón de Células no Pequeñas , Proteínas HSP70 de Choque Térmico , Neoplasias Pulmonares , Humanos , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas HSP70 de Choque Térmico/genética , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/genética , Autofagia/efectos de los fármacos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Línea Celular Tumoral , Proteínas Quinasas Activadas por AMP/metabolismo , Células A549 , Factores de Transcripción del Choque Térmico/metabolismo , Factores de Transcripción del Choque Térmico/genética , Sirolimus/farmacología , Apoptosis/efectos de los fármacos , Nucleósidos de Purina/farmacología , Isoxazoles , ResorcinolesRESUMEN
In this study, proximal fleximer nucleos(t)ide analogues of Bemnifosbuvir were synthesized and evaluated for their potential to serve as antiviral therapeutics. The final parent flex-nucleoside and ProTide modified flex-nucleoside analogues were tested against several viral families including flaviviruses, filoviruses, and coronaviruses. Modest activity against Zaire Ebola virus was observed at 30 µM for compound ProTide modified analogue. Neither compound exhibited activity for any of the other viruses tested. The parent flex-nucleoside analogue was screened for toxicity in CD-1 mice and showed no adverse effects up to 300 mg/kg, the maximum concentration tested.
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Antivirales , Antivirales/síntesis química , Antivirales/farmacología , Antivirales/química , Antivirales/farmacocinética , Animales , Ratones , Pruebas de Sensibilidad Microbiana , Relación Estructura-Actividad , Estructura Molecular , Relación Dosis-Respuesta a Droga , Humanos , Nucleósidos de Purina/síntesis química , Nucleósidos de Purina/farmacología , Nucleósidos de Purina/química , Nucleósidos de Purina/farmacocinéticaRESUMEN
Fulminant viral hepatitis (FH) represents a significant clinical challenge, with its pathogenesis not yet fully elucidated. Heat shock protein (HSP)70, a molecular chaperone protein with a broad range of cytoprotective functions, is upregulated in response to stress. However, the role of HSP70 in FH remains to be investigated. Notably, HSP70 expression is upregulated in the livers of coronavirus-infected mice and patients. Therefore, we investigated the mechanistic role of HSP70 in coronavirus-associated FH pathogenesis. FH was induced in HSP70-deficient (HSP70 KO) mice or in WT mice treated with the HSP70 inhibitor VER155008 when infected with the mouse hepatitis virus strain A59 (MHV-A59). MHV-A59-infected HSP70 KO mice exhibited significantly reduced liver damage and mortality. This effect was attributed to decreased infiltration of monocyte-macrophages and neutrophils in the liver of HSP70 KO mice, resulting in lower levels of inflammatory cytokines such as IL-1ß, TNFα, and IL-6, and a reduced viral load. Moreover, treatment with the HSP70 inhibitor VER155008 protected mice from MHV-A59-induced liver damage and FH mortality. In summary, HSP70 promotes coronavirus-induced FH pathogenesis by enhancing the infiltration of monocyte-macrophages and neutrophils and promoting the secretion of inflammatory cytokines. Therefore, HSP70 is a potential therapeutic target in viral FH intervention.
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Proteínas HSP70 de Choque Térmico , Hígado , Ratones Endogámicos C57BL , Ratones Noqueados , Virus de la Hepatitis Murina , Animales , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas HSP70 de Choque Térmico/genética , Virus de la Hepatitis Murina/patogenicidad , Ratones , Hígado/patología , Hígado/virología , Hígado/metabolismo , Citocinas/metabolismo , Humanos , Hepatitis Viral Animal/inmunología , Hepatitis Viral Animal/patología , Hepatitis Viral Animal/virología , Infecciones por Coronavirus/inmunología , Infecciones por Coronavirus/patología , Infecciones por Coronavirus/virología , Masculino , Macrófagos/inmunología , Nucleósidos de PurinaRESUMEN
The dynamic landscape of cellular nucleotides/nucleosides associated with RNA metabolism, particularly in diseases like cancer, has spurred intensive interest. Here, we report a robust stable isotope-diluted UHPLC-ESI-MS/MS method for accurate quantification of 12 purine ribonucleosides, including 10 methylated purine nucleosides. By the use of thermally decomposable ammonium bicarbonate (NH4HCO3) as a mobile phase additive for UHPLC-MS/MS detection, the ESI-MS/MS signal responses of these target compounds were enhanced by 1.7-24.5 folds. Noteworthily, three methylated guanosine isomers (m1G, m2G, and m7G) and two methylated adenosine isomers (m1A and m6A) that are indistinguishable directly by mass spectrometry were well resolved with optimal UHPLC separation. Combined with methanol extraction and solid-phase extraction (SPE) pretreatment, the method quantified intracellular concentrations of three modified nucleosides (Gm, m1G, and m2G), which would otherwise be undetectable because of significant suppression of their signals by the interfering cellular matrix. Nine purine nucleosides were simultaneously quantified in 293T cells, and their concentrations ranged by 4 orders of magnitude. Overall, the method presents high recovery rates over 90% for endogenous modified purine nucleosides in cultured cells, along with good precision, linearity, and LOD ranging from 0.30 fmol to 0.37 pmol per 5 × 105 cells. The developed UHPLC-MS/MS method holds potential for screening purine nucleosides as diagnostic and prognostic biomarkers and for quantifying purine epigenetic nucleosides post-cell metabolome analysis, thereby providing a valuable analytical tool for intracellular nucleoside quantification in future clinical research.
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Nucleósidos de Purina , Espectrometría de Masas en Tándem , Espectrometría de Masas en Tándem/métodos , Cromatografía Líquida de Alta Presión , Humanos , Nucleósidos de Purina/análisis , Nucleósidos de Purina/química , MetilaciónRESUMEN
Our recent work has uncovered a novel function of HSPA8 as an amyloidase, capable of dismantling the RHIM-containing protein fibrils to suppress necroptosis. However, the impact of HSPA8 inhibitors on cancer regression via necroptosis remains unexplored. In this study, we conducted a comprehensive investigation to assess the potential of HSPA8 inhibitors in enhancing necroptosis both in vitro and in vivo. Our findings indicate that pharmacologic inhibition of HSPA8, achieved either through VER (VER-155008) targeting the nucleotide binding domain or pifithrin-µ targeting the substrate binding domain of HSPA8, significantly potentiates necroptosis induced by diverse treatments in cellular assays. These inhibitors effectively disrupt the binding of HSPA8 to the RHIM protein, impeding its regulatory function on RHIM amyloid formation. Importantly, HSPA8 inhibitors significantly enhanced cancer cell sensitivity to microtubule-targeting agents (MTAs) in vitro, while reversing chemoresistance and facilitating tumor regression by augmenting necroptosis in vivo. Our findings suggest a promising therapeutic approach to cancer through necroptosis modulation via HSPA8 targeting, particularly in combination with MTA drugs for enhanced treatment efficacy.
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Proteínas del Choque Térmico HSC70 , Necroptosis , Neoplasias , Necroptosis/efectos de los fármacos , Humanos , Animales , Línea Celular Tumoral , Ratones , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Proteínas del Choque Térmico HSC70/metabolismo , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Ensayos Antitumor por Modelo de Xenoinjerto , Ratones Desnudos , Resistencia a Antineoplásicos/efectos de los fármacos , Nucleósidos de PurinaRESUMEN
Adenosine Deaminases Acting on RNA (ADARs) catalyze the deamination of adenosine to inosine in double-stranded RNA (dsRNA). ADARs' ability to recognize and edit dsRNA is dependent on local sequence context surrounding the edited adenosine and the length of the duplex. A deeper understanding of how editing efficiency is affected by mismatches, loops, and bulges around the editing site would aid in the development of therapeutic gRNAs for ADAR-mediated site-directed RNA editing (SDRE). Here, a SELEX (systematic evolution of ligands by exponential enrichment) approach was employed to identify dsRNA substrates that bind to the deaminase domain of human ADAR2 (hADAR2d) with high affinity. A library of single-stranded RNAs was hybridized with a fixed-sequence target strand containing the nucleoside analog 8-azanebularine that mimics the adenosine deamination transition state. The presence of this nucleoside analog in the library biased the screen to identify hit sequences compatible with adenosine deamination at the site of 8-azanebularine modification. SELEX also identified non-duplex structural elements that supported editing at the target site while inhibiting editing at bystander sites.
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Adenosina Desaminasa , Nucleósidos de Purina , Ribonucleósidos , Humanos , Adenosina , Adenosina Desaminasa/metabolismo , Secuencia de Bases , ARN Bicatenario , ARN Guía de Sistemas CRISPR-CasRESUMEN
Hypochlorous acid (HOCl) released from activated leukocytes plays a significant role in the human immune system, but is also implicated in numerous diseases due to its inappropriate production. Chlorinated nucleobases induce genetic changes that potentially enable and stimulate carcinogenesis, and thus have attracted considerable attention. However, their multiple halogenation sites pose challenges to identify them. As a good complement to experiments, quantum chemical computation was used to uncover chlorination sites and chlorinated products in this study. The results indicate that anion salt forms of all purine compounds play significant roles in chlorination except for adenosine. The kinetic reactivity order of all reaction sites in terms of the estimated apparent rate constant kobs-est (in M-1 s-1) is heterocyclic NH/N (102-107) > exocyclic NH2 (10-2-10) > heterocyclic C8 (10-5-10-1), but the order is reversed for thermodynamics. Combining kinetics and thermodynamics, the numerical simulation results show that N9 is the most reactive site for purine bases to form the main initial chlorinated product, while for purine nucleosides N1 and exocyclic N2/N6 are the most reactive sites to produce the main products controlled by kinetics and thermodynamics, respectively, and C8 is a possible site to generate the minor product. The formation mechanisms of biomarker 8-Cl- and 8-oxo-purine derivatives were also investigated. Additionally, the structure-kinetic reactivity relationship study reveals a good correlation between lg kobs-est and APT charge in all purine compounds compared to FED2 (HOMO), which proves again that the electrostatic interaction plays a key role. The results are helpful to further understand the reactivity of various reaction sites in aromatic compounds during chlorination.
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Nucleósidos , Contaminantes Químicos del Agua , Humanos , Nucleósidos/química , Halogenación , Dominio Catalítico , Nucleósidos de Purina , Ácido Hipocloroso/química , Cinética , Cloro/química , Contaminantes Químicos del Agua/químicaRESUMEN
Cyclic nucleotide binding domains (CNB) confer allosteric regulation by cAMP or cGMP to many signaling proteins, including PKA and PKG. PKA of phylogenetically distant Trypanosoma is the first exception as it is cyclic nucleotide-independent and responsive to nucleoside analogues (Bachmaier et al., 2019). Here, we show that natural nucleosides inosine, guanosine and adenosine are nanomolar affinity CNB ligands and activators of PKA orthologs of the important tropical pathogens Trypanosoma brucei, Trypanosoma cruzi, and Leishmania. The sequence and structural determinants of binding affinity, -specificity and kinase activation of PKAR were established by structure-activity relationship (SAR) analysis, co-crystal structures and mutagenesis. Substitution of two to three amino acids in the binding sites is sufficient for conversion of CNB domains from nucleoside to cyclic nucleotide specificity. In addition, a trypanosomatid-specific C-terminal helix (αD) is required for high affinity binding to CNB-B. The αD helix functions as a lid of the binding site that shields ligands from solvent. Selectivity of guanosine for CNB-B and of adenosine for CNB-A results in synergistic kinase activation at low nanomolar concentration. PKA pulldown from rapid lysis establishes guanosine as the predominant ligand in vivo in T. brucei bloodstream forms, whereas guanosine and adenosine seem to synergize in the procyclic developmental stage in the insect vector. We discuss the versatile use of CNB domains in evolution and recruitment of PKA for novel nucleoside-mediated signaling.
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AMP Cíclico , Nucleósidos de Purina , AMP Cíclico/metabolismo , Nucleósidos/farmacología , Regulación Alostérica , Nucleótidos Cíclicos , Guanosina , AdenosinaRESUMEN
BACKGROUND: CR6-interacting factor 1 (CRIF1), a multifunctional protein that affects mitochondrial function and cell senescence, plays a regulatory role in heart-related diseases. However, whether CRIF1 participates in myocardial senescence by regulating mitochondrial function remains unclear. METHODS: Doxorubicin (DOX)-induced C57BL/6 mice to construct mouse myocardial senescence model, and the myocardial function indicators including lactate dehydrogenase (LDH) and Creatine kinase isoform MB (CK-MB) were assessed. The expression of CRIF1 was detected by western blot. Myocardial pathological changes were examined by transthoracic echocardiography and haematoxylin and eosin (H&E) staining. Cell senescence was detected by SA-ß-gal staining. JC-1 staining was used to detect mitochondrial membrane potential. Biochemical kits were used to examine oxidative stress-related factors. Additionally, AC16 cardiomyocytes were treated with DOX to mimic the cellular senescence model in vitro. Cell activity was detected by cell counting kit-8 (CCK-8) assay. Co-immunoprecipitation (CO-IP) was used to verify the relationship between CRIF1 and peroxidasin (PXDN). RESULTS: The CRIF1 expression was significantly decreased in DOX-induced senescent mice and AC16 cells. Overexpression of CRIF1 significantly ameliorated DOX-induced myocardial dysfunction and myocardial senescence. Additionally, CRIF1 overexpression attenuated DOX-induced oxidative stress and myocardial mitochondrial dysfunction. Consistently, CRIF1 overexpression also inhibited DOX-induced oxidative stress and senescence in AC16 cells. Moreover, CRIF1 was verified to bind to PXDN and inhibited PXDN expression. The inhibitory effects of CRIF1 overexpression on DOX-induced oxidative stress and senescence in AC16 cells were partly abolished by PXDN expression. CONCLUSIONS: CRIF1 plays a protective role against DOX-caused mitochondrial dysfunction and myocardial senescence partly through downregulating PXDN.
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Desoxirribonucleósidos , Doxorrubicina , Enfermedades Mitocondriales , Nucleósidos de Purina , Ratones , Animales , Ratones Endogámicos C57BL , Doxorrubicina/toxicidad , Miocardio/metabolismo , Estrés Oxidativo , Miocitos Cardíacos/metabolismo , Enfermedades Mitocondriales/metabolismo , ApoptosisRESUMEN
Antimicrobial resistance is emerging in clinical strains of Clostridioides difficile. Ibezapolstat (IBZ) is a DNA polymerase IIIC inhibitor that has completed phase II clinical trials. IBZ has potent in vitro activity against wild-type, susceptible strains but its effect on C. difficile strains with reduced susceptibility to metronidazole (MTZ), vancomycin (VAN), or fidaxomicin (FDX) has not been tested. The primary objective of this study was to test the antibacterial properties of IBZ against multidrug-resistant C. difficile strains. The in vitro activity, bactericidal, and time-kill activity of IBZ versus comparators were evaluated against 100 clinical strains of which 59 had reduced susceptibility to other C. difficile antibiotics. Morphologic changes against a multidrug resistance strain were visualized by light and scanning electron microscopy. The overall IBZ MIC50/90 values (µg/mL) for evaluated C. difficile strains were 4/8, compared with 2/4 for VAN, 0.5/1 for FDX, and 0.25/4 for MTZ. IBZ MIC50/90 values did not differ based on non-susceptibility to antibiotic class or number of classes to which strains were non-susceptible. IBZ bactericidal activity was similar to the minimum inhibitory concentration (MIC) and maintained in wild-type and non-susceptible strains. Time-kill assays against two laboratory wild-type and two clinical non-susceptible strains demonstrated sustained IBZ activity despite reduced killing by comparator antibiotics for IBZ and VAN non-susceptible strains. Microscopy visualized increased cell lengthening and cellular damage in multidrug-resistant strains exposed to IBZ sub-MIC concentrations. This study demonstrated the potent antibacterial activity of IBZ against a large collection of C. difficile strains including multidrug-resistant strains. This study highlights the therapeutic potential of IBZ against multidrug-resistant strains of C. difficile.
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Antiinfecciosos , Clostridioides difficile , Infecciones por Clostridium , Nucleósidos de Purina , Humanos , Clostridioides , Infecciones por Clostridium/tratamiento farmacológico , Infecciones por Clostridium/microbiología , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Antiinfecciosos/farmacología , Vancomicina/farmacología , Vancomicina/uso terapéutico , Metronidazol/farmacología , Metronidazol/uso terapéutico , Fidaxomicina/farmacología , Fidaxomicina/uso terapéutico , Pruebas de Sensibilidad MicrobianaRESUMEN
This study evaluated 15 lactic acid bacteria with a focus on their ability to degrade inosine and hypo-xanthine-which are the intermediates in purine metabolism-for the management of hyperuricemia and gout. After a preliminary screening based on HPLC, Lactiplantibacillus plantarum CR1 and Lactiplantibacillus pentosus GZ1 were found to have the highest nucleoside degrading rates, and they were therefore selected for further characterization. S. thermophilus IDCC 2201, which possessed the hpt gene encoding hypoxanthine-guanine phosphoribosyltransferase (HGPRT) and exhibited purine degradation, was also selected for further characterization. These three selected strains were examined in terms of their probiotic effect on lowering serum uric acid in a Sprague-Dawley (SD) rat model of potassium oxonate (PO)-induced hyperuricemia. Among these three strains, the level of serum uric acid was most reduced by S. thermophilus IDCC 2201 (p < 0.05). Further, analysis of the microbiome showed that administration of S. thermophlilus IDCC 2201 led to a significant difference in gut microbiota composition compared to that in the group administered with PO-induced hyperuricemia. Moreover, intestinal short-chain fatty acids (SCFAs) were found to be significantly increased. Altogether, the results of this work indicate that S. thermophilus IDCC 2201 lowers uric acid levels by degrading purine-nucleosides and also restores intestinal flora and SCFAs, ultimately suggesting that S. thermophilus IDCC 2201 is a promising candidate for use as an adjuvant treatment in patients with hyperuricemia.
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Hiperuricemia , Nucleósidos de Purina , Ratas , Animales , Humanos , Nucleósidos de Purina/metabolismo , Ácido Úrico , Hiperuricemia/metabolismo , Nucleósidos , Streptococcus thermophilus , Ratas Sprague-Dawley , XantinaRESUMEN
Covering: 2019 to 2023Nucleoside analogues represent one of the most important classes of small molecule pharmaceuticals and their therapeutic development is successfully established within oncology and for the treatment of viral infections. However, there are currently no nucleoside analogues in clinical use for the management of bacterial infections. Despite this, a significant number of clinically recognised nucleoside analogues are known to possess some antibiotic activity, thereby establishing a potential source for new therapeutic discovery in this area. Furthermore, given the rise in antibiotic resistance, the discovery of new clinical candidates remains an urgent global priority and natural product-derived nucleoside analogues may also present a rich source of discovery space for new modalities. This Highlight, covering work published from 2019 to 2023, presents a current perspective surrounding the synthesis of natural purine nucleoside antibiotics. By amalgamating recent efforts from synthetic chemistry with advances in biosynthetic understanding and the use of recombinant enzymes, prospects towards different structural classes of purines are detailed.
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Antibacterianos , Nucleósidos de Purina , Antibacterianos/química , Antibacterianos/síntesis química , Antibacterianos/farmacología , Nucleósidos de Purina/química , Nucleósidos de Purina/síntesis química , Nucleósidos de Purina/farmacología , Productos Biológicos/química , Productos Biológicos/farmacología , Productos Biológicos/síntesis química , Estructura Molecular , HumanosRESUMEN
The synthesis and antiproliferative evaluation of novel d-glucopyranuronamide-containing nucleosides is described. Based on our previously reported anticancer d-glucuronamide-based nucleosides, new analogues comprising N/O-dodecyl or N-propargyl substituents at the glucuronamide unit and anomerically-N-linked 2-acetamido-6-chloropurine, 6-chloropurine or 4-(6-chloropurinyl)methyl triazole motifs were synthesized in 4-6 steps starting from acetonide-protected glucofuranurono-6,3-lactone. The methodologies were based on the access to N-substituted glycopyranuronamide precursors, namely 1,2-O-acetyl derivatives or glucuronoamidyl azides for further nucleobase N-glycosylation or 1,3-dipolar cycloaddition with N9 - and N7 -propargyl-6-chloropurines, respectively. N-Propargyl glucuronamide-based N9 -purine nucleosides were converted into (triazolyl)methyl amide-6,6-linked pseudodisaccharide nucleosides via cycloaddition with methyl 6-azido-glucopyranoside. A CuI/Amberlyst A-21 catalytic system employed in the cycloaddition reactions also effected conversion into 6-dimethylaminopurine nucleosides. Antiproliferative evaluation in chronic myeloid leukemia (K562) and breast cancer (MCF-7) cells revealed significant effects exhibited by the synthesized monododecylated purine-containing nucleosides. A N-propargyl 3-O-dodecyl glucuronamide derivative comprising a N9 -ß-linked 6-chloropurine moiety was the most active compound against MCF-7 cells (GI50 =11.9â µM) while a related α-(purinyl)methyltriazole nucleoside comprising a N7 -linked 6-chloropurine moiety exhibited the highest activity against K562 cells (GI50 =8.0â µM). Flow cytometry and immunoblotting analysis of apoptosis-related proteins in K562 cells treated with the N-propargyl 3-O-dodecyl glucuronamide-based N9 -linked 6-chloropurine nucleoside indicated that it acts via apoptosis induction.
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Amidas , Nucleósidos , Humanos , Nucleósidos/farmacología , Amidas/farmacología , Nucleósidos de Purina , GlucuronatosRESUMEN
Malaria remains a major public health threat for billions of people worldwide. Infection with obligate intracellular, unicellular parasites from the genus Plasmodium causes malaria. Plasmodium falciparum causes the deadliest form of human malaria. Plasmodium parasites are purine auxotrophic. They rely on purine import from the host red blood cell cytoplasm via equilibrative nucleoside transporters to supply substrates to the purine salvage pathway. We previously developed a high throughput screening assay to identify inhibitors of the P. falciparum Equilibrative Nucleoside Transporter Type 1 (PfENT1). Screening a small molecule library identified PfENT1 inhibitors that blocked proliferation of P. falciparum parasites in in vitro culture. The goal of the current work was to validate a high-resolution model of PfENT1 predicted by the AlphaFold protein structure prediction program. We superimposed the predicted PfENT1 structure on the human homologue structure, hENT1, and developed a structure-based sequence alignment. We mutated the residues in PfENT1 aligned with and flanking the residues in hENT1 that interact with the purine analog, nitrobenzylthioinosine (NBMPR). Mutation of the PfENT1 residues Q135, D287, and R291 that are predicted to form hydrogen bonds to purine nucleosides eliminated purine and pyrimidine transport function in various yeast-based growth and radiolabeled substrate uptake assays. Mutation of two flanking residues, W53 and S290, also resulted in inactive protein. Mutation of L50 that forms hydrophobic interactions with the purine nucleobase reduced transport function. Based on our results the AlphaFold predicted structure for PfENT1 may be useful in guiding medicinal chemistry efforts to improve the potency of our PfENT1 inhibitors.
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Malaria Falciparum , Malaria , Proteínas Portadoras de Nucleobases, Nucleósidos, Nucleótidos y Ácidos Nucleicos , Parásitos , Animales , Humanos , Nucleósidos de Purina/metabolismo , Parásitos/metabolismo , Proteínas Portadoras de Nucleobases, Nucleósidos, Nucleótidos y Ácidos Nucleicos/metabolismo , Malaria Falciparum/parasitología , Proteínas de Transporte de Membrana/metabolismo , Saccharomyces cerevisiae/genética , Tranportador Equilibrativo 1 de NucleósidoRESUMEN
Why does protein kinase A respond to purine nucleosides in certain pathogens, but not to the cyclic nucleotides that activate this kinase in most other organisms?
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Leishmania donovani , Trypanosoma brucei brucei , Ligandos , Fosfotransferasas/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Nucleósidos de Purina/metabolismoRESUMEN
In the final step of cytokinin biosynthesis, the main pathway is the elimination of a ribose-phosphate moiety from the cytokinin nucleotide precursor by phosphoribohydrolase, an enzyme encoded by a gene named LONELY GUY (LOG). This reaction accounts for most of the cytokinin supply needed for regulating plant growth and development. In contrast, the LOG-independent pathway, in which dephosphorylation and deribosylation sequentially occur, is also thought to play a role in cytokinin biosynthesis, but the gene entity and physiological contribution have been elusive. In this study, we profiled the phytohormone content of chromosome segment substitution lines of Oryza sativa and searched for genes affecting the endogenous levels of cytokinin ribosides by quantitative trait loci analysis. Our approach identified a gene encoding an enzyme that catalyzes the deribosylation of cytokinin nucleoside precursors and other purine nucleosides. The cytokinin/purine riboside nucleosidase 1 (CPN1) we identified is a cell wall-localized protein. Loss-of-function mutations (cpn1) were created by inserting a Tos17-retrotransposon that altered the cytokinin composition in seedling shoots and leaf apoplastic fluid. The cpn1 mutation also abolished cytokinin riboside nucleosidase activity in leaf extracts and attenuated the trans-zeatin riboside-responsive expression of cytokinin marker genes. Grain yield of the mutants declined due to altered panicle morphology under field-grown conditions. These results suggest that the cell wall-localized LOG-independent cytokinin activating pathway catalyzed by CPN1 plays a role in cytokinin control of rice growth. Our finding broadens our spatial perspective of the cytokinin metabolic system.
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Oryza , Oryza/genética , Citocininas/genética , Nucleósidos de Purina , N-Glicosil Hidrolasas/genética , Nucleósidos , Pared Celular/genéticaRESUMEN
In alternate organic synthesis, biocatalysis using enzymes provides a more stereoselective and cost-effective approach. Synthesis of unnatural nucleosides by nucleoside base exchange reactions using nucleoside-metabolizing enzymes has previously shown that the 5-position recognition of pyrimidine bases on nucleoside substrates is loose and can be used to introduce functional molecules into pyrimidine nucleosides. Here we explored the incorporation of purine pseudo bases into nucleosides by the base exchange reaction of pyrimidine nucleoside phosphorylase (PyNP), demonstrating that an imidazole five-membered ring is an essential structure for the reaction. In the case of benzimidazole, the base exchange proceeded to give the deoxyribose form in 96 % yield, and the ribose form in 23 % yield. The reaction also proceeded with 1H-imidazo[4,5-b]phenazine, a benzimidazole analogue with an additional ring, although the yield of nucleoside was only 31 %. Docking simulations between 1H and imidazo[4,5-b]phenazine nucleoside and the active site of PyNP (PDB 1BRW) supported our observation that 1H-imidazo[4,5-b]phenazine can be used as a substrate by PyNP. Thus, the enzymatic substitution reaction using PyNP can be used to incorporate many purine pseudo bases and benzimidazole derivatives with various functional groups into nucleoside structures, which have potential utility as diagnostic or therapeutic agents.
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Nucleósidos , Purinas , Nucleósidos/química , Bencimidazoles , Nucleósidos de Purina , Purina-Nucleósido Fosforilasa/metabolismoRESUMEN
Fluorescent nucleobase analogues (FBAs) are established tools for studying oligonucleotide structure, dynamics and interactions, and have recently also emerged as an attractive option for labeling RNA-based therapeutics. A recognized drawback of FBAs, however, is that they typically require excitation in the UV region, which for imaging in biological samples may have disadvantages related to phototoxicity, tissue penetration, and out-of-focus photobleaching. Multiphoton excitation has the potential to alleviate these issues and therefore, in this work, we characterize the multiphoton absorption properties and detectability of the highly fluorescent quadracyclic adenine analogue 2CNqA as a ribonucleotide monomer as well as incorporated, at one or two positions, into a 16mer antisense oligonucleotide (ASO). We found that 2CNqA has a two-photon absorption cross section that, among FBAs, is exceptionally high, with values of σ2PA(700 nm) = 5.8 GM, 6.8 GM, and 13 GM for the monomer, single-, and double-labelled oligonucleotide, respectively. Using fluorescence correlation spectroscopy, we show that the 2CNqA has a high 2P brightness as the monomer and when incorporated into the ASO, comparing favorably to other FBAs. We furthermore demonstrate the usefulness of the 2P imaging mode for improving detectability of 2CNqA-labelled ASOs in live cells.
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Colorantes Fluorescentes , Oligonucleótidos , Colorantes Fluorescentes/química , Espectrometría de Fluorescencia/métodos , Nucleósidos de Purina , Adenina/químicaRESUMEN
5'-18O labeled RNA oligos are important probes to investigate the mechanism of 2'-O-transphosphorylation reactions. Here we describe a general and efficient synthetic approach to the phosphoramidite derivatives of 5'-18O labeled nucleosides starting from the corresponding commercially available 5'-O-DMT protected nucleosides. Using this method, we prepared 5'-18O-guanosine phosphoramidite in 8 steps (13.2% overall yield), 5'-18O-adenosine phosphoramidite in 9 steps (10.1% overall yield) and 5'-18O-2'-deoxyguanosine phosphoramidite in 6 steps (12.8% overall yield). These 5'-18O labeled phosphoramidites can be incorporated into RNA oligos by solid phase synthesis for determination of heavy atom isotope effects in RNA 2'-O-transphosphorylation reactions.
Asunto(s)
Nucleósidos , Nucleósidos de Purina , ARN , Compuestos OrganofosforadosRESUMEN
Purine scaffolds constitute a starting point for the synthesis of numerous chemotherapeutics used in treating cancer, viruses, parasites, as well as bacterial and fungal infections. In this work, we synthesized a group of guanosine analogues containing an additional five-membered ring and a sulfur atom at the C-9 position. The spectral, photophysical, and biological properties of the synthesized compounds were investigated. The spectroscopic studies revealed that a combination of the thiocarbonyl chromophore and the tricyclic structure of guanine analogues shifts the absorption region above 350 nm, allowing for selective excitation when present in biological systems. Unfortunately, due to the low fluorescence quantum yield, this process cannot be used to monitor the presence of these compounds in cells. The synthesized compounds were evaluated for their effect on the viability of human cervical carcinoma (HeLa) and mouse fibroblast (NIH/3T3) cells. It was found that all of them display anticancer activity. In vitro studies were preceded by in silico ADME and PASS analyses, which confirmed that the designed compounds are promising candidates for anticancer agents.