RESUMEN

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.

Asunto(s)

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 , Resorcinoles

RESUMEN

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.

Asunto(s)

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/farmacocinética

RESUMEN

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.

Asunto(s)

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 , Humanos

RESUMEN

A number of purine arabinosides containing chiral amino acid amides at the C6 position of the purine were synthesized using a transglycosylation reaction with recombinant E. coli nucleoside phosphorylases. Arsenolysis of 2-chloropurine ribosides with chiral amino acid amides at C6 was used for the enzymatic synthesis, and the reaction equilibrium shifted towards the synthesis of arabinonucleosides. The synthesized nucleosides were shown to be resistant to the action of E. coli adenosine deaminase. The antiproliferative activity of the synthesized nucleosides was studied on human acute myeloid leukemia cell line U937. Among all the compounds, the serine derivative exhibited an activity level (IC50 = 16 µM) close to that of Nelarabine (IC50 = 3 µM) and was evaluated as active.

Asunto(s)

Escherichia coli , Nucleósidos de Purina , Humanos , Nucleósidos de Purina/farmacología , Escherichia coli/metabolismo , Aminoácidos , Nucleósidos/química , Arabinonucleósidos

RESUMEN

BACKGROUND: A new family of purine nucleoside cholinesterase inhibitors was disclosed by us, with potency and selectivity over acetylcholinesterase or butyrylcholinesterase controlled by tuning structural and stereochemical features of nucleosides with perbenzylated glycosyl moieties. OBJECTIVE: Design, synthesis, and biological evaluation of new purine nucleosides were used to investigate glycon protecting group pattern required for anticholinesterase activity and selectivity. METHODS: Regioselective chemistry to introduce methyl/benzyl groups in glycon donors and Nglycosylation was used to acquire the target nucleosides. Evaluation of their biological potential and selectivity as cholinesterase inhibitors was performed. RESULTS: Synthetic strategies chosen resulted in high glycon donor's overall yield and regio- and stereoselectivity was found in N-glycosylation reaction. Some of the new nucleosides are cholinesterase inhibitors and selectivity for butyrylcholinesterase was also achieved. CONCLUSION: N-glycosylation reaction was stereoselective for the ß-anomers while regioselectivity was achieved for the N9 isomers when glycon positions 2 and 3 were methylated. Cholinesterase inhibition was found when the 2,3-di-O-benzyl-4-O-methyl pattern is present in the sugar moiety. Amongst the new compounds, the two most promising ones showed micromolar inhibition (mixed inhibition), being one of them selective for butyrylcholinesterase inhibition.

Asunto(s)

Butirilcolinesterasa , Inhibidores de la Colinesterasa , Inhibidores de la Colinesterasa/química , Butirilcolinesterasa/metabolismo , Nucleósidos de Purina/farmacología , Acetilcolinesterasa/metabolismo , Nucleósidos/química , Relación Estructura-Actividad

RESUMEN

The purine-derived signaling molecules c-di-AMP and (p)ppGpp control mecA/PBP2a-mediated ß-lactam resistance in methicillin-resistant Staphylococcus aureus (MRSA) raise the possibility that purine availability can control antibiotic susceptibility. Consistent with this, exogenous guanosine and xanthosine, which are fluxed through the GTP branch of purine biosynthesis, were shown to significantly reduce MRSA ß-lactam resistance. In contrast, adenosine (fluxed to ATP) significantly increased oxacillin resistance, whereas inosine (which can be fluxed to ATP and GTP via hypoxanthine) only marginally increased oxacillin susceptibility. Furthermore, mutations that interfere with de novo purine synthesis (pur operon), transport (NupG, PbuG, PbuX) and the salvage pathway (DeoD2, Hpt) increased ß-lactam resistance in MRSA strain JE2. Increased resistance of a nupG mutant was not significantly reversed by guanosine, indicating that NupG is required for guanosine transport, which is required to reduce ß-lactam resistance. Suppressor mutants resistant to oxacillin/guanosine combinations contained several purine salvage pathway mutations, including nupG and hpt. Guanosine significantly increased cell size and reduced levels of c-di-AMP, while inactivation of GdpP, the c-di-AMP phosphodiesterase negated the impact of guanosine on ß-lactam susceptibility. PBP2a expression was unaffected in nupG or deoD2 mutants, suggesting that guanosine-induced ß-lactam susceptibility may result from dysfunctional c-di-AMP-dependent osmoregulation. These data reveal the therapeutic potential of purine nucleosides, as ß-lactam adjuvants that interfere with the normal activation of c-di-AMP are required for high-level ß-lactam resistance in MRSA. IMPORTANCE The clinical burden of infections caused by antimicrobial resistant (AMR) pathogens is a leading threat to public health. Maintaining the effectiveness of existing antimicrobial drugs or finding ways to reintroduce drugs to which resistance is widespread is an important part of efforts to address the AMR crisis. Predominantly, the safest and most effective class of antibiotics are the ß-lactams, which are no longer effective against methicillin-resistant Staphylococcus aureus (MRSA). Here, we report that the purine nucleosides guanosine and xanthosine have potent activity as adjuvants that can resensitize MRSA to oxacillin and other ß-lactam antibiotics. Mechanistically, exposure of MRSA to these nucleosides significantly reduced the levels of the cyclic dinucleotide c-di-AMP, which is required for ß-lactam resistance. Drugs derived from nucleotides are widely used in the treatment of cancer and viral infections highlighting the clinical potential of using purine nucleosides to restore or enhance the therapeutic effectiveness of ß-lactams against MRSA and potentially other AMR pathogens.

Asunto(s)

Staphylococcus aureus Resistente a Meticilina , Nucleósidos de Purina/metabolismo , Nucleósidos de Purina/farmacología , Proteínas Bacterianas/metabolismo , Antibacterianos/farmacología , Antibacterianos/metabolismo , Oxacilina/farmacología , beta-Lactamas/farmacología , Monobactamas/metabolismo , Monobactamas/farmacología , Guanosina/metabolismo , Guanosina/farmacología , Adenosina Trifosfato/metabolismo , Guanosina Trifosfato/metabolismo , Pruebas de Sensibilidad Microbiana , Proteínas de Unión a las Penicilinas/genética , Proteínas de Unión a las Penicilinas/metabolismo , Resistencia betalactámica/genética

RESUMEN

Purine nucleosides represent an interesting group of nitrogen heterocycles, showing a wide range of biological effects. In this study, we designed and synthesized a series of 6,9-disubstituted and 2,6,9-trisubstituted purine ribonucleosides via consecutive nucleophilic aromatic substitution, glycosylation, and deprotection of the ribofuranose unit. We prepared eight new purine nucleosides bearing unique adamantylated aromatic amines at position 6. Additionally, the ability of the synthesized purine nucleosides to form stable host-guest complexes with ß-cyclodextrin (ß-CD) was confirmed using nuclear magnetic resonance (NMR) and mass spectrometry (ESI-MS) experiments. The in vitro antiproliferative activity of purine nucleosides and their equimolar mixtures with ß-CD was tested against two types of human tumor cell line. Six adamantane-based purine nucleosides showed an antiproliferative activity in the micromolar range. Moreover, their effect was only slightly suppressed by the presence of ß-CD, which was probably due to the competitive binding of the corresponding purine nucleoside inside the ß-CD cavity.

Asunto(s)

Adamantano , beta-Ciclodextrinas , Humanos , Adamantano/farmacología , Nucleósidos de Purina/farmacología , Nucleósidos de Purina/metabolismo , beta-Ciclodextrinas/farmacología , Línea Celular Tumoral , Nucleósidos/farmacología , Nucleósidos/química

RESUMEN

The transient receptor potential cation channel, subfamily M, members 6 and 7 (TRPM6 and TRPM7) are homologous membrane proteins encompassing cation channel units fused to cytosolic serine/threonine-protein kinase domains. Clinical studies and experiments with animal disease models suggested that selective inhibition of TRPM6 and TRPM7 currents might be beneficial for subjects with immune and cardiovascular disorders, tumours and other pathologies, but the suitable pharmacological toolkit remains underdeveloped. The present study identified small synthetic molecules acting specifically on the channel moieties of TRPM6 and TRPM7. Using electrophysiological analysis in conjunction with Ca2+ imaging, we show that iloperidone and ifenprodil inhibit the channel activity of recombinant TRPM6 with IC50 values of 0.73 and 3.33 µM, respectively, without an impact on the TRPM7 channel. We also found that VER155008 suppresses the TRPM7 channel with an IC50 value of 0.11 µM but does not affect TRPM6. Finally, the effects of iloperidone and VER155008 were found to be suitable for blocking native endogenous TRPM6 and TRPM7 in a collection of mouse and human cell models. Hence, the identification of iloperidone, ifenprodil, and VER155008 allows for the first time to selectively manipulate TRPM6 and TRPM7 currents.

Asunto(s)

Canales Catiónicos TRPM , Animales , Humanos , Isoxazoles/farmacología , Magnesio/metabolismo , Ratones , Piperidinas/farmacología , Proteínas Serina-Treonina Quinasas , Nucleósidos de Purina/farmacología , Canales Catiónicos TRPM/efectos de los fármacos , Canales Catiónicos TRPM/metabolismo , Canales de Potencial de Receptor Transitorio/efectos de los fármacos , Canales de Potencial de Receptor Transitorio/metabolismo

RESUMEN

Leishmaniasis causes high mortality and morbidity in tropical and subtropical regions of Africa, Asia, the Americas and southern Europe, and is characterized by diverse clinical manifestations. As a neglected tropical disease, limited resources are allocated for antileishmanial drug discovery. The Leishmania parasite is deficient in de novo purine synthesis, and therefore acquires purines from the host and processes these using a purine salvage pathway. By making use of purine transport systems and interfering with this salvage pathway, purine (nucleoside) analogues might exert a selective detrimental impact on its growth and survival. In vitro screening of an in-house purine nucleoside library and analogue synthesis afforded the 6-methyl-7-(2-pyridyl)-7-deazapurine ribonucleoside analogue 18 as a promising hit. Optimization of the 7-substituent afforded 31 and 32 which displayed potent activity against wild-type and resistant L. infantum, intracellular amastigote and extracellular promastigote forms, and favorable selectivity versus primary mouse macrophages (Mφ) and MRC-5 cells. Encouraged by the favorable in vitro metabolic stability of 32, an in vivo study was performed using an early curative L. infantum hamster model. When orally administrated at 50 mg/kg once daily (s.i.d) for 10 days, 32 was devoid of side effects, however, it only poorly reduced amastigote burdens in the major target organs.

Asunto(s)

Antiprotozoarios , Leishmania , Leishmaniasis , Purinas , Ribonucleósidos , Animales , Antiprotozoarios/farmacología , Antiprotozoarios/uso terapéutico , Cricetinae , Leishmania/efectos de los fármacos , Leishmania/metabolismo , Leishmaniasis/tratamiento farmacológico , Ratones , Nucleósidos/farmacología , Nucleósidos/uso terapéutico , Nucleósidos de Purina/farmacología , Nucleósidos de Purina/uso terapéutico , Purinas/farmacología , Purinas/uso terapéutico , Ribonucleósidos/farmacología , Ribonucleósidos/uso terapéutico

RESUMEN

The 5',8-cyclo-2'-deoxypurines (cdPus) affect the DNA structure. When these bulky structures are a part of clustered DNA lesions (CDL), they affect the repair of the other lesions within the cluster. Mitochondria are crucial for cell survival and have their own genome, hence, are highly interesting in the context of CDL repair. However, no studies are exploring this topic. Here, the initial stages of mitochondrial base excision repair (mtBER) were considered-the strand incision and elongation. The repair of a single lesion (apurinic site (AP site)) accompanying the cdPu within the double-stranded CDL has been investigated for the first time. The type of cdPu, its diastereomeric form, and the interlesion distance were taken into consideration. For these studies, the established experimental model of short oligonucleotides (containing AP sites located ≤7 base pairs to the cdPu in both directions) and mitochondrial extracts of the xrs5 cells were used. The obtained results have shown that the presence of cdPus influenced the processing of an AP site within the CDL. Levels of strand incision and elongation were higher for oligos containing RcdA and ScdG than for those with ScdA and RcdG. Investigated stages of mtBER were more efficient for DNA containing AP sites located on 5'-end side of cdPu than on its 3'-end side. In conclusion, the presence of cdPus in mtDNA structure may affect mtBER (processing the second mutagenic lesion within the CDL). As impaired repair processes may lead to serious biological consequences, further studies concerning the mitochondrial repair of CDL are highly demanded.

Asunto(s)

Daño del ADN , Reparación del ADN , ADN Mitocondrial/metabolismo , Oligonucleótidos , Nucleósidos de Purina , Animales , Células CHO , Cricetulus , Oligonucleótidos/química , Oligonucleótidos/farmacología , Nucleósidos de Purina/química , Nucleósidos de Purina/farmacología

RESUMEN

Gastric cancer (GC) has one of the highest tumor incidences worldwide. Heat shock protein 70 (HSP70) is highly expressed and plays a critical role in the occurrence, progression, metastasis, poor prognosis, and drug resistance of GC. However, the underlying mechanisms of HSP70 are not clear. To explore the regulatory role of HSP70 in GC, we performed cell counting kit-8 (CCK-8) and EdU staining assays to assess cell proliferation; immunohistochemistry and western blot analyses to assess protein expression; coimmunoprecipitation (Co-IP) assays to assess interactions between two proteins; and immunofluorescence to assess protein expression and localization. HSP70 was highly expressed in clinical samples from patients with GC and indicated a poor prognosis. HSP70 inhibition enhanced the sensitivity of GC cells to thermochemotherapy. Furthermore, we found that S phase kinase-associated protein 2 (Skp2) was highly expressed in GC and correlated with HSP70 in array data from The Cancer Genome Atlas (TCGA). Importantly, HSP70 inhibition promoted Skp2 degradation. Skp2 overexpression abrogated HSP70 inhibition-induced cell cycle arrest, suggesting that the role of HSP70 in GC depends on Skp2 expression. Our results illustrate a possible regulatory mechanism of HSP70 and may provide a therapeutic strategy for overcoming resistance to thermochemotherapy.

Asunto(s)

Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas Quinasas Asociadas a Fase-S/química , Proteínas Quinasas Asociadas a Fase-S/metabolismo , Neoplasias Gástricas/metabolismo , Regulación hacia Arriba , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Progresión de la Enfermedad , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Masculino , Oxaliplatino/farmacología , Pronóstico , Estabilidad Proteica , Nucleósidos de Purina/farmacología , Regulación hacia Arriba/efectos de los fármacos

RESUMEN

To find efficient and broad-spectrum viral agents, a series of purine nucleoside derivatives containing sulfa ethylamine moieties was designed and synthesized, and their antiviral activities against tobacco mosaic virus (TMV), cucumber mosaic virus (CMV), and potato virus Y (PVY) were evaluated. Some target compounds displayed good antiviral activities. Among them, compound 3 showed excellent protective activity against CMV and PVY with 50% effective concentration values (EC50) of 137 and 209 µg/mL, respectively, which were better than that of the control agent ningnanmycin (508 and 431 µg/mL). Moreover, the EC50 value of compound 3 for the inactivating activity against TMV was 48 µg/mL, which was better than that of ningnanmycin (88 µg/mL). In addition, compound 3 not only destroyed the structure of the TMV virus but also had a good interaction with the coat protein of the TMV virus. Therefore, compound 3 may further destroy the structure of the virus by binding to the coat protein of the TMV virus, thereby weakening the infectivity of the virus.

Asunto(s)

Nucleósidos de Purina , Virus del Mosaico del Tabaco , Antivirales/farmacología , Diseño de Fármacos , Etilaminas , Nucleósidos , Nucleósidos de Purina/farmacología , Relación Estructura-Actividad

RESUMEN

Kinetoplastid parasites are the causative agents of neglected tropical diseases with an unmet medical need. These parasites are unable to synthesize the purine ring de novo, and therefore rely on purine salvage to meet their purine demand. Evaluating purine nucleoside analogs is therefore an attractive strategy to identify antikinetoplastid agents. Several anti-Trypanosoma cruzi and anti-Trypanosoma brucei 7-deazapurine nucleosides were previously discovered, with the removal of the 3'-hydroxyl group resulting in a significant boost in activity. In this work we therefore decided to assess the effect of the introduction of a 3'-fluoro substituent in 7-deazapurine nucleosides on the anti-kinetoplastid activities. Hence, we synthesized two series of 3'-deoxy-3'-fluororibofuranosyl and 3'-deoxy-3'-fluoroxylofuranosyl nucleosides comprising 7-deazaadenine and -hypoxanthine bases and assayed these for antiparasitic activity. Several analogs with potent activity against T. cruzi and T. brucei were discovered, indicating that a fluorine atom in the 3'-position is a promising modification for the discovery of antiparasitic nucleosides.

Asunto(s)

Nucleósidos de Purina/química , Purinas/química , Tripanocidas/síntesis química , Línea Celular , Supervivencia Celular/efectos de los fármacos , Humanos , Nucleósidos de Purina/síntesis química , Nucleósidos de Purina/farmacología , Purinas/síntesis química , Purinas/farmacología , Relación Estructura-Actividad , Tripanocidas/química , Tripanocidas/farmacología , Trypanosoma brucei brucei/efectos de los fármacos , Trypanosoma cruzi/efectos de los fármacos

RESUMEN

In the present work, 103 novel acyclic nucleosides were designed, synthesized, and evaluated for their anticancer activities in vitro and in vivo. The structure-activity relationship (SAR) studies revealed that most target compounds inhibited the growth of colon cancer cells in vitro, of which 3-(6-chloro-9H-purin-9-yl)dodecan-1-ol (9b) exhibited the most potent effect against the HCT-116 and SW480 cells with IC50 values of 0.89 and 1.15 µM, respectively. Furthermore, all of the (R)-configured acyclic nucleoside derivatives displayed more potent anticancer activity compared to their (S)-counterparts. Mechanistic studies revealed that compound 9b triggered apoptosis in the cancer cell lines via depolarization of the mitochondrial membrane and effectively inhibited colony formation. Importantly, compound 9b inhibited the growth of the SW480 xenograft in a mouse model with low systemic toxicity. These results indicated that acyclic nucleoside compounds are viable as potent and effective anticancer agents, and compound 9b may serve as a promising lead compound that merits further attention in future anticancer drug discovery.

Asunto(s)

Antineoplásicos/uso terapéutico , Neoplasias/tratamiento farmacológico , Nucleósidos de Purina/uso terapéutico , Animales , Antineoplásicos/síntesis química , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Diseño de Fármacos , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Humanos , Masculino , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Ratones Endogámicos BALB C , Mitocondrias/efectos de los fármacos , Estructura Molecular , Nucleósidos de Purina/síntesis química , Nucleósidos de Purina/farmacología , Relación Estructura-Actividad , Ensayos Antitumor por Modelo de Xenoinjerto

RESUMEN

High levels or long periods of stress have been shown to negatively impact cell homeostasis, including with respect to abnormalities in domestic animal reproduction, which are typically activated through the hypothalamus-pituitary-adrenal axis, in which corticotropin-releasing hormone (CRH) and heat shock protein 70 (HSP70) are involved. In addition, CRH has been reported to inhibit pituitary gonadotrophin synthesis, and HSP70 is expressed in the pituitary gland. The aim of this study was to determine whether HSP70 was involved in regulating gonadotrophin synthesis and secretion by mediating the CRH pathway in the porcine pituitary gland. Our results showed that HSP70 was highly expressed in the porcine pituitary gland, with over 90% of gonadotrophic cells testing HSP70 positive. The results of functional studies demonstrated that the HSP70 inducer decreased FSH and LH levels in cultured porcine primary pituitary cells, whereas an HSP70 inhibitor blocked the negative effect of CRH on gonadotrophin synthesis and secretion. Furthermore, our results demonstrated that HSP70 inhibited gonadotrophin synthesis and secretion by blocking GnRH-induced SMAD3 phosphorylation, which acts as the targeting molecule of HSP70, while CRH upregulated HSP70 expression through the PKC and ERK pathways. Collectively, these data demonstrate that HSP70 inhibits pituitary gonadotrophin synthesis and secretion by regulating the CRH signaling pathway and inhibiting SMAD3 phosphorylation, which are important for our understanding the mechanisms of the stress affects domestic animal reproductive functions.

Asunto(s)

Hormona Liberadora de Corticotropina/metabolismo , Gonadotropinas/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Transducción de Señal/fisiología , Proteína smad3/metabolismo , Porcinos/metabolismo , Animales , Células Cultivadas , Hormona Liberadora de Corticotropina/genética , Hormona Folículo Estimulante de Subunidad beta/genética , Hormona Folículo Estimulante de Subunidad beta/metabolismo , Regulación de la Expresión Génica , Gliceraldehído-3-Fosfato Deshidrogenasa (Fosforilante) , Gonadotropinas/genética , Hormona Luteinizante , Morfolinas/farmacología , Hipófisis/citología , Nucleósidos de Purina/farmacología , Piridinas/farmacología , Reacción en Cadena en Tiempo Real de la Polimerasa , Proteína smad3/genética , Urea/análogos & derivados , Urea/farmacología

RESUMEN

BACKGROUND: Pleural mesothelioma, a devastating asbestos-associated malignancy, urgently requires a novel effective therapy. Heat shock protein 70 (HSP70), which is synthesized in the cell response to protein damage, is expected to be a new target for antitumor treatment. In addition to its well-known protein refolding function, HSP70 regulates cell proliferation through different pathways, including PI3K/AKT/mTOR, and autophagy in malignant cells. In this study, we attempted to clarify the effects of VER-155008, an HSP70 inhibitor, on pleural mesothelioma. METHODS: Human pleural mesothelioma cell lines 211H, H2452 and H28 were cultured with VER-155008, and protein expression, cell proliferation, colony formation, cell cycle, synergistic effect with cisplatin, and autophagy induction were analyzed. RESULTS: In mesothelioma cell lines, VER-155008 (5.0 µM or more) inhibited cell growth and colony formation, accompanied by G1 cell cycle arrest. According to western blot analysis, VER-155008 reduced p-AKT expression. However, VER-155008 failed to show a synergistic effect with cisplatin on cell growth. Mesothelioma cells transfected with the novel plasmid pMRX-IP-GFP-LC3-RFP-LC3ΔG, which was developed for the quantitative and statistical estimation of macroautophagy, showed enhanced macroautophagy upon treatment with VER-155008 and gefitinib which is an EGFR-tyrosine kinase inhibitor. In addition, fetal bovine serum deprivation induced macroautophagy was further enhanced by VER-155008. CONCLUSIONS: On the basis of these results, functional HSP70 inhibition by VER-155008 suppressed cell growth in pleural mesothelioma cells, accompanied by enhanced macroautophagy. HSP70 inhibition is thus expected to become a new strategy for treating mesothelioma. KEY POINTS: Significant findings of the study In pleural mesothelioma cells, inhibition of HSP70 function by VER-155008 suppressed cell proliferation accompanied by induction of autophagy which was synergistically enhanced under the starvation condition, whereas gefitinib, an EGFR-TKI, did not show the same synergistic effect in autophagy. What this study adds The inhibition of HSP70 induced autophagy and suppressed cell proliferation in mesothelioma cells.

Asunto(s)

Proteínas HSP70 de Choque Térmico/metabolismo , Mesotelioma/tratamiento farmacológico , Neoplasias Pleurales/tratamiento farmacológico , Nucleósidos de Purina/uso terapéutico , Autofagia , Línea Celular Tumoral , Proliferación Celular , Humanos , Mesotelioma/patología , Neoplasias Pleurales/patología , Nucleósidos de Purina/farmacología , Transfección

RESUMEN

Methods for rapid and high-throughput screening of transcription in vitro to examine reaction conditions, enzyme mutants, promoter variants, and small molecule modulators can be extremely valuable tools. However, these techniques may be difficult to establish or inaccessible to many researchers. To develop a straightforward and cost-effective platform for assessing transcription in vitro, we used the "Broccoli" RNA aptamer as a direct, real-time fluorescent transcript readout. To demonstrate the utility of our approach, we screened the effect of common reaction conditions and components on bacteriophage T7 RNA polymerase (RNAP) activity using a common quantitative PCR instrument for fluorescence detection. Several essential conditions for in vitro transcription by T7 RNAP were confirmed with this assay, including the importance of enzyme and substrate concentrations, covariation of magnesium and nucleoside triphosphates, and the effects of several typical additives. When we used this method to assess all possible point mutants of a canonical T7 RNAP promoter, our results coincided well with previous reports. This approach should translate well to a broad variety of bacteriophage in vitro transcription systems and provides a platform for developing fluorescence-based readouts of more complex transcription systems in vitro.

Asunto(s)

Aptámeros de Nucleótidos/genética , Bioensayo , ARN Polimerasas Dirigidas por ADN/genética , ADN/genética , Reacción en Cadena de la Polimerasa/métodos , Proteínas Virales/genética , Secuencia de Aminoácidos , Aptámeros de Nucleótidos/química , Aptámeros de Nucleótidos/metabolismo , Secuencia de Bases , ADN/química , ADN/metabolismo , ARN Polimerasas Dirigidas por ADN/química , ARN Polimerasas Dirigidas por ADN/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Regulación de la Expresión Génica , Magnesio/química , Magnesio/farmacología , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Mutación Puntual , Regiones Promotoras Genéticas , Nucleósidos de Purina/química , Nucleósidos de Purina/farmacología , Nucleósidos de Pirimidina/química , Nucleósidos de Pirimidina/farmacología , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Cloruro de Sodio/química , Cloruro de Sodio/farmacología , Espectrometría de Fluorescencia , Espermidina/química , Espermidina/farmacología , Fracciones Subcelulares/metabolismo , Transcripción Genética , Proteínas Virales/química , Proteínas Virales/metabolismo

RESUMEN

Many cancers lack the expression of methylthioadenosine phosphorylase (MTAP). These cancers require adenylosuccinate synthetase (AdSS) for nucleic acid synthesis. By inhibiting adenylosuccinate synthetase, we potentially have a new therapeutic agent. Bisubstrate inhibitors were synthesized and evaluated against purified AdSS. The best activity was obtained with adenosine bearing a four-carbon linker that connects the N-formyl-N-hydroxy moiety to the 6-position of the purine nucleoside.

Asunto(s)

Diseño de Fármacos , Inhibidores Enzimáticos/farmacología , Nucleósidos de Purina/farmacología , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Humanos , Estructura Molecular , Nucleósidos de Purina/síntesis química , Nucleósidos de Purina/química , Purina-Nucleósido Fosforilasa

RESUMEN

AIMS: HSP70, a molecular chaperone, helps to maintain proteostasis. In muscle biology, however, evidence suggests HSP70 to have a more versatile range of functions, as genetic deletion of its inducible genes impairs Ca2+ handling, and consequently, cardiac and skeletal muscle contractility. Still, it is unknown whether HSP70 is involved in vascular reactivity, an intrinsic physiological mechanism of blood vessels. Therefore, we designed this study to test the hypothesis that proper vascular reactivity requires the assistance of HSP70. MAIN METHODS: We performed functional studies in a wire-myograph using thoracic aorta isolated from male Sprague Dawley rats. Experiments were conducted with and without an HSP70 inhibitor as well as in heat-stressed vessels. The expression levels of HSP70 were evaluated with Western blotting. NO and ROS levels were assessed with fluorescence microscopy. KEY FINDINGS: We report that blockade of HSP70 weakens contraction in response to phenylephrine (dose-response) in the aorta. Additionally, we demonstrated that inhibition of HSP70 affects the amplitude of the fast and of the slow components of the time-force curve. Corroborating these findings, we found that inhibition of HSP70, in vessels over-expressing this protein, partly rescues the contractile phenotype of aortic rings. Furthermore, we show that blockade of HSP70 facilitates relaxation in response to acetylcholine and clonidine without affecting the basal levels of NO and ROS. SIGNIFICANCE: Our work introduces an additional physiological role for HSP70, the assistance of vascular reactivity, which highlights this protein as a new player in vascular physiology, and therefore, uncovers a promising research avenue for vascular diseases.

Asunto(s)

Aorta Torácica/fisiología , Endotelio Vascular/fisiología , Proteínas HSP70 de Choque Térmico/fisiología , Músculo Liso Vascular/fisiología , Acetilcolina/farmacología , Animales , Aorta Torácica/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Endotelio Vascular/efectos de los fármacos , Proteínas HSP70 de Choque Térmico/agonistas , Proteínas HSP70 de Choque Térmico/antagonistas & inhibidores , Masculino , Músculo Liso Vascular/efectos de los fármacos , Técnicas de Cultivo de Órganos , Fenilefrina/farmacología , Nucleósidos de Purina/farmacología , Ratas , Ratas Sprague-Dawley , Vasoconstrictores/farmacología , Vasodilatadores/farmacología

RESUMEN

BACKGROUND: Infectious bursal disease (IBD) is a highly contagious infectious disease that causes severe immunosuppression and damage to the bursa of Fabricius in chickens. Several proteins involved in IBD virus (IBDV) infection, such as surface immunoglobulin M, integrin, annexin A2 and chicken heat shock protein 90, have been identified. However, the main protein that plays key roles in virus infection has not yet been confirmed. METHODS: DF-1 cell line was transfected with the pcDNA-VP2 plasmid and analyzed by immunofluorescence assay. The proteins reacted with VP2 of IBDV in DF-1 cells were pulldown with the monoclonal antibody and identified by mass spectrometry. Heat shock cognate protein 70 (HSC70), one of these proteins, was selected to be investigated in the function in IBDV infection by specific antibody and its inhibitor. RESULTS: The DF-1 cell line was transfected with the pcDNA-VP2 plasmid, and expression of IBDV VP2 in DF-1 cells was confirmed by immunofluorescence assays. Heat shock cognate protein 70 (HSC70) was one of the proteins identified by coimmunoprecipitation using a monoclonal antibody (2H11) against VP2 and mass spectrometry analysis. IBDV infection in DF-1 cells was strongly inhibited by both an anti-HSC70 antibody and a HSC70 inhibitor (VER155008). CONCLUSION: These results suggest that HSC70 may be an essential factor for IBDV infection.

Asunto(s)

Fibroblastos/virología , Proteínas del Choque Térmico HSC70/genética , Virus de la Enfermedad Infecciosa de la Bolsa/genética , Virus de la Enfermedad Infecciosa de la Bolsa/patogenicidad , Animales , Anticuerpos Antivirales , Línea Celular , Pollos/virología , Técnica del Anticuerpo Fluorescente , Proteínas del Choque Térmico HSC70/inmunología , Virus de la Enfermedad Infecciosa de la Bolsa/efectos de los fármacos , Enfermedades de las Aves de Corral/virología , Nucleósidos de Purina/farmacología , Proteínas Estructurales Virales/genética