RESUMEN
Chemodynamic therapy (CDT) is a novel antitumor strategy that employs Fenton or Fenton-like reactions to generate highly toxic hydroxyl radical (OHâ¢) from hydrogen peroxide (H2O2) for inducing tumor cell death. However, the antitumor efficacy of the CDT strategy is harshly limited by the redox homeostasis of tumor cells; especially the OH ⢠is easily scavenged by glutathione (GSH) and the intracellular H2O2 level is insufficient in the tumor cells. Herein, we propose the Mn2+-menadione (also known as vitamin K3, MK3) cascade biocatalysis strategy to disrupt the redox homeostasis of tumor cells and induce a OH⢠storm, resulting in enhanced CDT effect. A nanoliposome encapsulating Mn-MK3 (Mn-MK3@LP) was prepared for the treatment of hepatic tumors in this study. After Mn-MK3@LPs were taken up by tumor cells, menadione could facilitate the production of intracellular H2O2 via redox cycling, and further the cytotoxic OH ⢠burst was induced by Mn2+-mediated Fenton-like reaction. Moreover, high-valent manganese ions were reduced by GSH and the depletion of GSH further disrupted the redox homeostasis of tumor cells, thus achieving synergistically enhanced CDT. Overall, both cellular and animal experiments confirmed that the Mn-MK3@LP cascade biocatalysis nanoliposome exhibited excellent biosafety and tumor suppression efficacy. This study may provide deep insights for developing novel CDT-based strategies for tumor therapy.
Asunto(s)
Glutatión , Peróxido de Hidrógeno , Radical Hidroxilo , Vitamina K 3 , Animales , Radical Hidroxilo/química , Radical Hidroxilo/metabolismo , Humanos , Ratones , Glutatión/metabolismo , Glutatión/química , Vitamina K 3/química , Vitamina K 3/farmacología , Biocatálisis , Línea Celular Tumoral , Manganeso/química , Oxidación-Reducción/efectos de los fármacos , Ratones Endogámicos BALB C , Liposomas/química , Ensayos Antitumor por Modelo de Xenoinjerto , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/metabolismo , Ratones Desnudos , Células Hep G2 , Antineoplásicos/farmacología , Antineoplásicos/química , Nanopartículas/química , HierroRESUMEN
Understanding the mechanisms of drug action in malarial parasites is crucial for the development of new drugs to combat infection and to counteract drug resistance. Proteomics is a widely used approach to study host-pathogen systems and to identify drug protein targets. Plasmodione is an antiplasmodial early-lead drug exerting potent activities against young asexual and sexual blood stages inâ vitro with low toxicity to host cells. To elucidate its molecular mechanisms, an affinity-based protein profiling (AfBPP) approach was applied to yeast and P. falciparum proteomes. New (pro-) AfBPP probes based on the 3-benz(o)yl-6-fluoro-menadione scaffold were synthesized. With optimized conditions of both photoaffinity labeling and click reaction steps, the AfBPP protocol was then applied to a yeast proteome, yielding 11 putative drug-protein targets. Among these, we found four proteins associated with oxidoreductase activities, the hypothesized type of targets for plasmodione and its metabolites, and other proteins associated with the mitochondria. In Plasmodium parasites, the MS analysis revealed 44 potential plasmodione targets that need to be validated in further studies. Finally, the localization of a 3-benzyl-6-fluoromenadione AfBPP probe was studied in the subcellular structures of the parasite at the trophozoite stage.
Asunto(s)
Antimaláricos , Plasmodium falciparum , Proteómica , Vitamina K 3 , Antimaláricos/farmacología , Antimaláricos/química , Plasmodium falciparum/efectos de los fármacos , Vitamina K 3/farmacología , Vitamina K 3/química , Vitamina K 3/metabolismo , Proteínas Protozoarias/metabolismo , Etiquetas de Fotoafinidad/química , Etiquetas de Fotoafinidad/farmacología , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/efectos de los fármacos , Sondas Moleculares/química , Sondas Moleculares/farmacología , Proteoma/análisis , Proteoma/metabolismo , Estructura MolecularRESUMEN
To minimize background interference in electrochemical enzymatic biosensors employing electron mediators, it is essential for the electrochemical oxidation of electroactive interfering species (ISs), such as ascorbic acid (AA), to proceed slowly, and for the redox reactions between electron mediators and ISs to occur at a low rate. In this study, we introduce a novel combination of a working electrode and an electron mediator that effectively mitigates interference effects. Compared to commonly used electrodes such as Au, glassy carbon, and indium tin oxide (ITO), boron-doped diamond (BDD) electrodes demonstrate significantly lower anodic current (i.e., lower background levels) in the presence of AA. Additionally, menadione (MD) exhibits notably slower reactivity with AA compared to other electron mediators such as Ru(NH3)63+, 4-amino-1-naphthol, and 1,4-naphthoquinone, primarily due to the lower formal potential of MD compared to AA. This synergistic combination of BDD electrode and MD is effectively applied in three biosensors: (i) glucose detection using electrochemical-enzymatic (EN) redox cycling, (ii) glucose detection using electrochemical-enzymatic-enzymatic (ENN) redox cycling, and (iii) lactate detection using ENN redox cycling. Our developed approach significantly outperforms the combination of ITO electrode and MD in minimizing IS interference. Glucose in artificial serum can be detected with detection limits of ~ 20 µM and ~ 3 µM in EN and ENN redox cycling, respectively. Furthermore, lactate in human serum can be detected with a detection limit of ~ 30 µM. This study demonstrates sensitive glucose and lactate detection with minimal interference, eliminating the need for (bio)chemical agents to remove interfering species.
Asunto(s)
Boro , Diamante , Técnicas Electroquímicas , Electrodos , Glucosa , Ácido Láctico , Vitamina K 3 , Diamante/química , Vitamina K 3/química , Boro/química , Glucosa/análisis , Glucosa/química , Ácido Láctico/análisis , Ácido Láctico/química , Técnicas Biosensibles , Electrones , Humanos , Oxidación-ReducciónRESUMEN
Azo dyes are important to various industries such as textile industries. However, these dyes are known to comprise toxic, mutagenic, and carcinogenic representatives. Several approaches have already been employed to mitigate the problem such as the use of enzymes. Azoreductases have been well-studied in its capability to reduce azo dyes. AzoRo from Rhodococcus opacus 1CP has been found to be accepting only methyl red as a substrate, surmising that the enzyme may have a narrow active site. To determine the active site configuration of AzoRo at atomic level and identify the key residues involved in substrate binding and enzyme specificity, we have determined the crystal structure of holo-AzoRo and employed a rational design approach to generate AzoRo variants. The results reported here show that AzoRo has a different configuration of the active site when compared with other bacterial NAD(P)H azoreductases, having other key residues playing a role in the substrate binding and restricting the enzyme activity towards different azo dyes. Moreover, it was observed that AzoRo has only about 50% coupling yield to methyl red and p-benzoquinone - giving rise to the possibility that NADH oxidation still occurs even during catalysis. Results also showed that AzoRo is more active and more efficient towards quinones (about four times higher than methyl red).
Asunto(s)
Compuestos Azo/química , Mezclas Complejas/química , NADH NADPH Oxidorreductasas/metabolismo , NAD/metabolismo , Quinonas/química , Rhodococcus/química , Catálisis , Dominio Catalítico , Clonación Molecular , Cristalización , Cinética , NADH NADPH Oxidorreductasas/genética , Unión Proteica , Conformación Proteica , Especificidad por Sustrato , Vitamina K 3/químicaRESUMEN
Menadione (VK3) is used as a powerful inducer of cellular reactive oxygen species (ROS) for many years and displays the high anti-cancer activities in vivo. Recently, the development of mitochondria-targeted drugs has been more and more appreciated. Here, the thirteen derivatives of VK3 were synthesized, which could localize in mitochondria by the triphenylphosphonium (TPP) cation or the nitrogen-based cation. The results of cytotoxicity from six human cancer cell lines showed that the targeted compounds T1-T13 displayed higher activity than VK3 with the average IC50 value around 1 µM. The results of cytotoxicity indicated that the substitutes on C-2, the linear alkyl chains on C-3 and cation moiety all could affect the cytotoxicity. The mechanistic studies showed that five representative compounds (T2, T3, T5, T8 and T13) could localize in cellular mitochondria, elicit ROS burst and collapse mitochondrial membrane potential (ΔΨm), leading to cytochrome C release and apoptosis in MGC-803 cells. Particularly, they could obviously inhibit mitochondrial thioredoxin reductase TrxR2 expression, thus leading to aggravate cellular oxidative stress.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Tiorredoxina Reductasa 2/antagonistas & inhibidores , Vitamina K 3/farmacología , Antineoplásicos/síntesis química , Antineoplásicos/química , Cationes/síntesis química , Cationes/química , Cationes/farmacología , Línea Celular , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Mitocondrias/metabolismo , Estructura Molecular , Relación Estructura-Actividad , Tiorredoxina Reductasa 2/metabolismo , Vitamina K 3/síntesis química , Vitamina K 3/químicaRESUMEN
Although podophyllotoxin (POD) demonstrates high efficiency to inhibit various cancers, its clinic application is limited to poor bioavailability. Nanoparticles derived from homodimeric prodrugs with high drug loading potential are emerging as promising nanomedicines. However, complete intracellular drug release remains a major hindrance to the use of homodimeric prodrugs-based nanomedicine. We sought to develop a reactive oxygen species (ROS) responsive POD dimeric prodrug by incorporating vitamin K3 (VK3) and Pluronic F127 to synthesize a spheroid nanoparticle (PTV-NPs). PTV-NPs with high POD content could release drugs under the ROS enrichment microenvironment in cancer cells. The released VK3 could produce abundant ROS selectively in tumor cells catalyzed by the overexpressed NAD(P)H: quinone oxidoreductase-1 (NQO1) enzyme. In turn, the resultant high ROS concentration promoted the conversion of POD dimeric prodrug to POD monomer, thereby achieving the selective killing of cancer cells with weak system toxicity. In vitro and in vivo studies consistently confirmed that PTV-NPs exhibit high drug loading potential and upstanding bioavailability. They are also effectively internalized by tumor cells, induce abundant intracellular ROS generation, and have high tumor-specific cytotoxicity. This ROS-responsive dimeric prodrug nanoplatform characterized by selective self-amplification drug release may hold promise in the field of antitumor drug delivery.
Asunto(s)
Antineoplásicos Fitogénicos/administración & dosificación , Neoplasias/patología , Podofilotoxina/administración & dosificación , Podofilotoxina/farmacología , Profármacos/administración & dosificación , Profármacos/farmacología , Animales , Antineoplásicos Fitogénicos/farmacología , Línea Celular Tumoral , Química Farmacéutica , Relación Dosis-Respuesta a Droga , Liberación de Fármacos , Estabilidad de Medicamentos , Femenino , Humanos , Concentración de Iones de Hidrógeno , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , NAD(P)H Deshidrogenasa (Quinona)/metabolismo , NADP/metabolismo , Nanopartículas/química , Podofilotoxina/farmacocinética , Poloxámero/química , Polímeros/química , Profármacos/farmacocinética , Especies Reactivas de Oxígeno/metabolismo , Microambiente Tumoral/fisiología , Vitamina K 3/química , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Ficus deltoidea var. deltoidea is used as traditional medicine for diabetes, inflammation, and nociception. However, the antimutagenic potential and cytoprotective effects of this plant remain unknown. In this study, the mutagenic and antimutagenic activities of F. deltoidea aqueous extract (FDD) on both Salmonella typhimurium TA 98 and TA 100 strains were assessed using Salmonella mutagenicity assay (Ames test). Then, the cytoprotective potential of FDD on menadione-induced oxidative stress was determined in a V79 mouse lung fibroblast cell line. The ferric-reducing antioxidant power (FRAP) assay was conducted to evaluate FDD antioxidant capacity. Results showed that FDD (up to 50 mg/mL) did not exhibit a mutagenic effect on either TA 98 or TA 100 strains. Notably, FDD decreased the revertant colony count induced by 2-aminoanthracene in both strains in the presence of metabolic activation (p < 0.05). Additionally, pretreatment of FDD (50 and 100 µg/mL) demonstrated remarkable protection against menadione-induced oxidative stress in V79 cells significantly by decreasing superoxide anion level (p < 0.05). FDD at all concentrations tested (12.5-100 µg/mL) exhibited antioxidant power, suggesting the cytoprotective effect of FDD could be partly attributed to its antioxidant properties. This report highlights that F. deltoidea may provide a chemopreventive effect on mutagenic and oxidative stress inducers.
Asunto(s)
Antimutagênicos/química , Antioxidantes/química , Ficus/metabolismo , Extractos Vegetales/química , Animales , Aniones , Línea Celular , Cricetulus , Diabetes Mellitus , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Glutatión , Ratones , Mutagénesis/efectos de los fármacos , Pruebas de Mutagenicidad , Mutágenos , Estrés Oxidativo , Salmonella typhimurium/efectos de los fármacos , Sales de Tetrazolio/química , Tiazoles/química , Vitamina K 3/química , AguaRESUMEN
After the emergence of the pandemic, repurposed drugs have been considered as a quicker way of finding potential antiviral agents. SARS-CoV-2 3CLpro is essential for processing the viral polyproteins into mature non-structural proteins, making it an attractive target for developing antiviral agents. Here we show that Vitamin K3 screened from the FDA-Approved Drug Library containing an array of 1,018 compounds has potent inhibitory activity against SARS-CoV-2 3CLpro with the IC50 value of 4.78 ± 1.03 µM, rather than Vitamin K1, K2 and K4. Next, the time-dependent inhibitory experiment was carried out to confirm that Vitamin K3 could form the covalent bond with SARS-CoV-2 3CLpro. Then we analyzed the structure-activity relationship of Vitamin K3 analogues and identified 5,8-dihydroxy-1,4-naphthoquinone with 9.8 times higher inhibitory activity than Vitamin K3. Further mass spectrometric analysis and molecular docking study verified the covalent binding between Vitamin K3 or 5,8-dihydroxy-1,4-naphthoquinone and SARS-CoV-2 3CLpro. Thus, our findings provide valuable information for further optimization and design of novel inhibitors based on Vitamin K3 and its analogues, which may have the potential to fight against SARS-CoV-2.
Asunto(s)
Proteasas 3C de Coronavirus , Inhibidores de Cisteína Proteinasa/química , SARS-CoV-2/enzimología , Vitamina K 3 , Proteasas 3C de Coronavirus/antagonistas & inhibidores , Proteasas 3C de Coronavirus/química , Inhibidores de Cisteína Proteinasa/uso terapéutico , Humanos , Vitamina K 3/análogos & derivados , Vitamina K 3/química , Tratamiento Farmacológico de COVID-19RESUMEN
The utilization of a multifunctional bioactive molecule functionalized electrospun dressing in tissue repair and regenerative function is a prominent therapeutic strategy for preparing efficient biomaterials to promote chronic wound healing. Designing robust and highly efficient antibacterial agents in resistance against microbes and bacterial infections is a key challenge for accelerating diabetic wound healing until today. In this study, we developed a vitamin K3 carnosine peptide (VKC)-laden silk fibroin electrospun scaffold (SF-VKC) for diabetic wound healing. The structural confirmation of synthesized VKC was characterized by 1H NMR, 13C NMR, electrospray ionization mass spectrometry (ESI-MS), and attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy analysis, and the cell viability of VKC was evaluated by the CCK-8 assay in HFF1 and NIH 3T3 cells. VKC shows excellent cell viability on both cell lines, and the VKC and SF-VKC electrospun mats exhibited excellent antibacterial activity against both Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli and Pseudomonas aeruginosa) bacteria. Prepared SF and SF-VKC fibrous mats were well characterized, and the SF-VKC nanofiber mat presented good biodegradability, adhesiveness, unique mechanical property, expedient water uptake property, sustained drug release, and excellent biocompatibility for chronic wound healing. The in vitro tissue engineering study depicted excellent cell migration and cell-cell interaction in the NIH 3T3 cells over the VKC-impregnated silk fibroin (SF-VKC) mat. A higher population of cell migration was observed in cells' denuded area (scratched region) compared to the native SF fibrous mat. Interestingly, our results demonstrated that the prepared VKC-impregnated SF mat had potentially promoted the STZ-induced diabetic wound healing in a shorter period than the pure SF mat. Thus, obtained in vitro and in vivo outcomes suggest that the VKC-laden SF electrospun fibrous mat could be a better and inexpensive fibrous antibacterial biomaterial to elicit earlier re-epithelialization and efficient matrix remodeling for accelerating chronic infected wound reconstruction in skin diabetic wound healing applications.
Asunto(s)
Antibacterianos/administración & dosificación , Carnosina/administración & dosificación , Diabetes Mellitus Experimental/tratamiento farmacológico , Fibroínas/administración & dosificación , Vitamina K 3/administración & dosificación , Cicatrización de Heridas/efectos de los fármacos , Animales , Antibacterianos/química , Vendajes , Carnosina/química , Línea Celular , Supervivencia Celular/efectos de los fármacos , Diabetes Mellitus Experimental/complicaciones , Liberación de Fármacos , Escherichia coli/efectos de los fármacos , Escherichia coli/crecimiento & desarrollo , Fibroblastos/efectos de los fármacos , Fibroínas/química , Humanos , Masculino , Ratones , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/crecimiento & desarrollo , Ratas Sprague-Dawley , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/crecimiento & desarrollo , Ingeniería de Tejidos , Andamios del Tejido , Vitamina K 3/químicaRESUMEN
BACKGROUND: The current approach to reducing bacterial contamination in blood transfusion products is through detection or pathogen reduction methods, some of which utilize ultraviolet (UV) light photosensitizers. A small number of photosensitizers are being used as single agents in combination with UV light, but their efficacy can be limited against some pathogens. Benzophenone (BP) and vitamins B1, B6, and K3 have been identified as effective UVA photosensitizers for inactivation of bacteria. We evaluated whether combining pairs of photosensitizers in this group would have synergistic bactericidal effects on Gram-negative and Gram-positive bacteria. STUDY DESIGN AND METHODS: Bacteria species of Escherichia coli, Bacillus cereus, Staphylococcus aureus, and Klebsiella pneumoniae were mixed with 0 to 100 mM concentrations of photosensitizers and exposed to UVA irradiation at 18 J/cm2 to assess their bactericidal effects. RESULTS: Single photosensitizers irradiated with UVA produced a range of bactericidal activity. When combined in pairs, all demonstrated some synergistic bactericidal effects with up to 4-log reduction above the sum of activities of individual molecules in the pair against bacteria in plasma. Photosensitizer pairs with BP had the highest synergism across all bacteria. With vitamin K3 in the pair, synergism was evident for Gram-positive but not for Gram-negative bacteria. Vitamin B1 and vitamin B6 had the least synergism. These results indicate that a combination approach with multiple photosensitizers may extend effectiveness of pathogen reduction in plasma. CONCLUSIONS: Combining photosensitizers in pathogen reduction methods could improve bactericidal efficacy and lead to use of lower concentrations of photosensitizers to reduce toxicities and unwanted side effects.
Asunto(s)
Antibacterianos/efectos de la radiación , Benzofenonas/efectos de la radiación , Bacterias Gramnegativas/efectos de los fármacos , Bacterias Grampositivas/efectos de los fármacos , Fármacos Fotosensibilizantes/efectos de la radiación , Tiamina/efectos de la radiación , Rayos Ultravioleta , Vitamina B 6/efectos de la radiación , Vitamina K 3/efectos de la radiación , Absorción de Radiación , Antibacterianos/farmacología , Benzofenonas/química , Benzofenonas/farmacología , Sinergismo Farmacológico , Bacterias Gramnegativas/efectos de la radiación , Bacterias Grampositivas/efectos de la radiación , Humanos , Estructura Molecular , Fotoquímica , Fármacos Fotosensibilizantes/farmacología , Tiamina/química , Tiamina/farmacología , Vitamina B 6/química , Vitamina B 6/farmacología , Vitamina K 3/química , Vitamina K 3/farmacologíaRESUMEN
Redox-active substances and their combinations, such as of quinone/ascorbate and in particular menadione/ascorbate (M/A; also named Apatone®), attract attention with their unusual ability to kill cancer cells without affecting the viability of normal cells as well as with the synergistic anticancer effect of both molecules. So far, the primary mechanism of M/A-mediated anticancer effects has not been linked to the mitochondria. The aim of our study was to clarify whether this "combination drug" affects mitochondrial functionality specifically in cancer cells. Studies were conducted on cancer cells (Jurkat, Colon26, and MCF7) and normal cells (normal lymphocytes, FHC, and MCF10A), treated with different concentrations of menadione, ascorbate, and/or their combination (2/200, 3/300, 5/500, 10/1000, and 20/2000 µM/µM of M/A). M/A exhibited highly specific and synergistic suppression on cancer cell growth but without adversely affecting the viability of normal cells at pharmacologically attainable concentrations. In M/A-treated cancer cells, the cytostatic/cytotoxic effect is accompanied by (i) extremely high production of mitochondrial superoxide (up to 15-fold over the control level), (ii) a significant decrease of mitochondrial membrane potential, (iii) a decrease of the steady-state levels of ATP, succinate, NADH, and NAD+, and (iv) a decreased expression of programed cell death ligand 1 (PD-L1)-one of the major immune checkpoints. These effects were dose dependent. The inhibition of NQO1 by dicoumarol increased mitochondrial superoxide and sensitized cancer cells to M/A. In normal cells, M/A induced relatively low and dose-independent increase of mitochondrial superoxide and mild oxidative stress, which seems to be well tolerated. These data suggest that all anticancer effects of M/A result from a specific mechanism, tightly connected to the mitochondria of cancer cells. At low/tolerable doses of M/A (1/100-3/300 µM/µM) attainable in cancer by oral and parenteral administration, M/A sensitized cancer cells to conventional anticancer drugs, exhibiting synergistic or additive cytotoxicity accompanied by impressive induction of apoptosis. Combinations of M/A with 13 anticancer drugs were investigated (ABT-737, barasertib, bleomycin, BEZ-235, bortezomib, cisplatin, everolimus, lomustine, lonafarnib, MG-132, MLN-2238, palbociclib, and PI-103). Low/tolerable doses of M/A did not induce irreversible cytotoxicity in cancer cells but did cause irreversible metabolic changes, including: (i) a decrease of succinate and NADH, (ii) depolarization of the mitochondrial membrane, and (iii) overproduction of superoxide in the mitochondria of cancer cells only. In addition, M/A suppressed tumor growth in vivo after oral administration in mice with melanoma and the drug downregulated PD-L1 in melanoma cells. Experimental data suggest a great potential for beneficial anticancer effects of M/A through increasing the sensitivity of cancer cells to conventional anticancer therapy, as well as to the immune system, while sparing normal cells. We hypothesize that M/A-mediated anticancer effects are triggered by redox cycling of both substances, specifically within dysfunctional mitochondria. M/A may also have a beneficial effect on the immune system, making cancer cells "visible" and more vulnerable to the native immune response.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Mitocondrias/efectos de los fármacos , Neoplasias/tratamiento farmacológico , Animales , Apoptosis/efectos de los fármacos , Ácido Ascórbico/administración & dosificación , Ácido Ascórbico/química , Proliferación Celular/efectos de los fármacos , Quimioterapia Adyuvante , Femenino , Humanos , Células Jurkat , Células MCF-7 , Melanoma Experimental/tratamiento farmacológico , Melanoma Experimental/metabolismo , Melanoma Experimental/patología , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Mitocondrias/metabolismo , Mitocondrias/patología , Neoplasias/metabolismo , Neoplasias/patología , Especies Reactivas de Oxígeno/metabolismo , Células Tumorales Cultivadas , Vitamina K 3/administración & dosificación , Vitamina K 3/química , Vitaminas/administración & dosificación , Vitaminas/químicaRESUMEN
Thioredoxin reductase (TrxR) is one class of the most important antioxidant selenoproteins and is involved in regulating tumor genesis and progression. It has been reported that naphthoquinones can target and inhibit TrxR1 activity therefore produce reactive oxygen species (ROS) mediated by TrxR1, resulting into cellular redox imbalance and making the naphthoquinone compounds to become potential antitumor chemotherapy drugs. The purpose of this work is to explore the interaction between TrxR1 and menadione using biochemical and mass-spectrometric (MS) analyses, to further reveal the detailed mechanisms of TrxR1-mediated naphthoquinone reduction and inhibition of TrxR1 by naphthoquinone compounds. Using the site-directed mutagenesis and recombinantly expressed TrxR1 variants, we measured the steady-state kinetic parameters of menadione reduction mediated by TrxR1 and its variants, performed the inhibition analysis of menadione on TrxR1 activity, and eventually identified the interaction between menadione and TrxR1 through MS analysis. We found that Sec-to-Cys mutation at residue of 498 significantly enhanced the efficiency of TrxR1-mediated menadione reduction, though the Sec498 is capable to catalyze the menadione reduction, indicating that TrxR1-mediated menadione reduction is dominantly in a Se-independent manner. Mutation experiments showed that Cys498 is mainly responsible for menadione catalysis in comparison to Cys497, while the N-terminal Cys64 is slightly stronger than Cys59 regarding the menadione reduction. LC-MS results detected that TrxR1 was arylated with one molecule of menadione, suggesting that menadione irreversibly modified the hyper-reactive Sec residue at the C-terminus of selenoprotein TrxR1. This study revealed that TrxR1 catalyzes the reduction of menadione in a Se-independent manner meanwhile its activity is irreversibly inhibited by menadione. Hereby it will be useful for the research and development of naphthoquinone anticancer drugs targeting TrxR1.
Asunto(s)
Tiorredoxina Reductasa 1 , Vitamina K 3 , Catálisis , Desarrollo de Medicamentos , Oxidación-Reducción , Tiorredoxina Reductasa 1/metabolismo , Vitamina K 3/química , Vitamina K 3/metabolismoRESUMEN
Epigenetics plays a vital role in regulating gene expression and determining the specific phenotypes of eukaryotic cells. Histone deacetylases (HDACs) are important epigenetic regulatory proteins effecting multiple biological functions. Particularly, HDAC6 has become a promising anti-cancer drug target because of its regulation of cell mobility, protein trafficking, degradation of misfolded proteins, cell growth, apoptosis, and metastasis. In this study, we identified one out of six vitamin K3 derivatives, VKT-2, as HDAC6 inhibitor using molecular docking and cell viability assays in HDAC6-overexpressing HuH-7 cancer cells. Microscale thermophoresis and HDAC6 enzymatic assays revealed that VKT-2 bound to HDAC6 and inhibited its function. We further identified its cytotoxic activity. VKT-2 hyperacetylated HDAC6 substrates and disturbed tubulin integrity leading to significant inhibition of tumor migration in both HuH-7 spheroids and U2OS-GFP-α-tubulin cells. Moreover, VKT-2 induced autophagic and apoptotic cell death in HuH-7, while aggresome formation was restrained after VKT-2 treatment. A HuH-7 cell-xenograft model in zebrafish larvae provided evidence that VKT-2 inhibited the tumor growth in vivo. To best of our knowledge, it is the first time to demonstrate that vitamin k3 derivatives (VKT-2) inhibits HDAC6 in solid tumor cells. These unique findings suggested that VKT-2 is a promising anti-cancer agent targeting HDAC6.
Asunto(s)
Apoptosis/efectos de los fármacos , Autofagia/efectos de los fármacos , Carcinoma Hepatocelular/tratamiento farmacológico , Histona Desacetilasa 6/antagonistas & inhibidores , Inhibidores de Histona Desacetilasas/farmacología , Neoplasias Hepáticas/tratamiento farmacológico , Vitamina K 3/farmacología , Animales , Apoptosis/fisiología , Autofagia/fisiología , Carcinoma Hepatocelular/metabolismo , Agregación Celular/efectos de los fármacos , Agregación Celular/fisiología , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Relación Dosis-Respuesta a Droga , Células HCT116 , Células HEK293 , Histona Desacetilasa 6/metabolismo , Inhibidores de Histona Desacetilasas/química , Inhibidores de Histona Desacetilasas/uso terapéutico , Humanos , Neoplasias Hepáticas/metabolismo , Células MCF-7 , Vitamina K 3/química , Vitamina K 3/uso terapéutico , Ensayos Antitumor por Modelo de Xenoinjerto/métodos , Pez CebraRESUMEN
The crystal structure of the enzyme previously characterized as a type-2 NADH:menaquinone oxidoreductase (NDH-2) from Thermus thermophilus has been solved at a resolution of 2.9â¯Å and revealed that this protein is, in fact, a coenzyme A-disulfide reductase (CoADR). Coenzyme A (CoASH) replaces glutathione as the major low molecular weight thiol in Thermus thermophilus and is maintained in the reduced state by this enzyme (CoADR). Although the enzyme does exhibit NADH:menadione oxidoreductase activity expected for NDH-2 enzymes, the specific activity with CoAD as an electron acceptor is about 5-fold higher than with menadione. Furthermore, the crystal structure contains coenzyme A covalently linked Cys44, a catalytic intermediate (Cys44-S-S-CoA) reduced by NADH via the FAD cofactor. Soaking the crystals with menadione shows that menadione can bind to a site near the redox active FAD, consistent with the observed NADH:menadione oxidoreductase activity. CoADRs from other species were also examined and shown to have measurable NADH:menadione oxidoreductase activity. Although a common feature of this family of enzymes, no biological relevance is proposed. The CoADR from T. thermophilus is a soluble homodimeric enzyme. Expression of the recombinant TtCoADR at high levels in E. coli results in a small fraction that co-purifies with the membrane fraction, which was used previously to isolate the enzyme wrongly identified as a membrane-bound NDH-2. It is concluded that T. thermophilus does not contain an authentic NDH-2 component in its aerobic respiratory chain.
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Coenzima A/metabolismo , NADH NADPH Oxidorreductasas/química , NADH NADPH Oxidorreductasas/metabolismo , Thermus thermophilus/enzimología , Coenzima A/química , Escherichia coli , Modelos Moleculares , Proteínas Recombinantes , Electricidad Estática , Vitamina K 3/química , Difracción de Rayos XRESUMEN
Heme oxygenase (HO) enzymes are involved in heme catabolism and several physiological functions. Among the different HO isoforms, HO-2 stands out for its neuroprotective properties and modulatory activity in male reproduction. However, unlike the HO-1 ligands, the potential therapeutic applications of HO-2 inhibitors/activators have not been extensively explored yet. Moreover, the physiological role of HO-2 is still unclear, mostly due to the lack of highly selective HO-2 chemical probes. To boost the interest on this intriguing target, the present review updates the knowledge on the structure-activity relationships of HO-2 inhibitors and activators, as well as their potential therapeutic applications. To the best of our knowledge, among HO-2 inhibitors, clemizole derivatives are the most selective HO-2 inhibitors reported so far (IC50 HO-1 >100⯵M, IC50 HO-2â¯=â¯3.4⯵M), while the HO-2 nonselective inhibitors described herein possess IC50 HO-2 valuesâ¯≤â¯10⯵M. Furthermore, the development of HO-2 activators, such as menadione analogues, helped to understand the critical moieties required for HO-2 activation. Recent advances in the potential therapeutic applications of HO-2 inhibitors/activators cover the fields of neurodegenerative, cardiovascular, inflammatory, and reproductive diseases further stimulating the interest towards this target.
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Bencimidazoles/farmacología , Inhibidores Enzimáticos/farmacología , Hemo Oxigenasa (Desciclizante)/antagonistas & inhibidores , Hemo Oxigenasa (Desciclizante)/metabolismo , Vitamina K 3/farmacología , Animales , Bencimidazoles/química , Inhibidores Enzimáticos/química , Humanos , Estructura Molecular , Vitamina K 3/químicaRESUMEN
NAD(P)H:quinone oxidoreductase 1 (NQO1) is a key enzyme providing cytoprotection from quinone species. In addition, it is expressed at high levels in many human tumors, such as breast cancer. Therefore, it is considered to be a potential target in cancer treatment. In order to detect intracellular NQO1 activity in MCF-7 aggregates as a cancer model, we present, in this study, a double-mediator system combined with large-scale integration (LSI)-based amperometric devices. This LSI device contained 20 × 20 Pt working electrodes with a 250 µm pitch for electrochemical imaging. In the detection system, menadione (MD) and [Fe(CN)6]3- were used. Since MD can diffuse into cells due to its hydrophobicity, it is reduced into menadiol by intracellular NQO1. The menadiol diffuses out of the cells and reduces [Fe(CN)6]3- of a hydrophilic mediator into [Fe(CN)6]4-. The accumulated [Fe(CN)6]4- outside the cells is electrochemically detected at 0.5 V in the LSI device. Using this strategy, the intracellular NQO1 activity of MCF-7 aggregates was successfully detected. The effect of rotenone, which is an inhibitor for Complex I, on NQO1 activity was also investigated. In addition, NQO1 and respiration activities were simultaneously imaged using the detection system that was further combined with electrochemicolor imaging. Thus, the double-mediator system was proven to be useful for evaluating intracellular redox activity of cell aggregates.
Asunto(s)
Técnicas Electroquímicas/métodos , Pruebas de Enzimas/métodos , Ferricianuros/química , NAD(P)H Deshidrogenasa (Quinona)/análisis , Vitamina K 3/metabolismo , Respiración de la Célula/fisiología , Técnicas Electroquímicas/instrumentación , Complejo I de Transporte de Electrón/antagonistas & inhibidores , Pruebas de Enzimas/instrumentación , Humanos , Células MCF-7 , NAD(P)H Deshidrogenasa (Quinona)/metabolismo , Imagen Óptica/instrumentación , Imagen Óptica/métodos , Oxidación-Reducción , Rotenona/farmacología , Vitamina K/análogos & derivados , Vitamina K/química , Vitamina K 3/químicaRESUMEN
Vitamin K family molecules-phylloquinone (K1), menaquinone (K2), and menadione (K3)-act as γ-glutamyl carboxylase (GGCX)-exclusive cofactors in their hydroquinone state, activating proteins of main importance for blood coagulation in the liver and for arterial calcification prevention and energy metabolism in extrahepatic tissues. Once GGCX is activated, vitamin K is found in the epoxide state, which is then recycled to quinone and hydroquinone states by vitamin K epoxide reductase (VKORC1). Nevertheless, little information is available concerning vitamin K1, K2, or K3 tissue distribution and preferential interactions towards VKORC1. Here we present a molecular modeling study of vitamin K1, menaquinones 4, 7 (MK4, MK7), and K3 structural interactions with VKORC1. VKORC1 was shown to tightly bind vitamins K1 and MK4 in the epoxide and quinone states, but not in the hydroquinone state; five VKORC1 residues were identified as crucial for vitamin K stabilization, and two other ones were essential for hydrogen bond formation. However, vitamin MK7 revealed shaky binding towards VKORC1, induced by hydrophobic tail interactions with the membrane. Vitamin K3 exhibited the lowest affinity with VKORC1 because of the absence of a hydrophobic tail, preventing structural stabilization by the enzyme. Enzymatic activity towards vitamins K1, MK4, MK7, and K3 was also evaluated by in vitro assays, validating our in silico predictions: VKORC1 presented equivalent activities towards vitamins K1 and MK4, but much lower activity with respect to vitamin MK7, and no activity towards vitamin K3. Our results revealed VKORC1's ability to recycle both phylloquinone and some menaquinones, and also highlighted the importance of vitamin K's hydrophobic tail size and membrane interactions.
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Vitamina K 1/química , Vitamina K 2/química , Vitamina K 3/química , Vitamina K Epóxido Reductasas/metabolismo , Sitios de Unión , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Modelos Moleculares , Pichia/metabolismo , Unión Proteica , Conformación Proteica , Vitamina K 1/metabolismo , Vitamina K 2/metabolismo , Vitamina K 3/metabolismo , Vitamina K Epóxido Reductasas/químicaRESUMEN
Patients with esophageal carcinoma (ESCA) have a poor prognosis and high mortality rate. Although standard therapies have had effect, there is an urgent requirement to develop novel options, as increasing drug tolerance has been identified in clinical practice. In the present study, differentially expressed genes (DEGs) of ESCA were identified in The Cancer Genome Atlas and GenotypeTissue Expression databases. Functional and proteinprotein interaction (PPI) analyses were performed. The Connectivity Map (CMAP) was selected to predict drugs for the treatment of ESCA, and their target genes were acquired from the Search Tool for Interactions of Chemicals (STITCH) by uploading the Simplified MolecularInput LineEntry System structure. Additionally, significant target genes and ESCAassociated hub genes were extracted using another PPI analysis, and the corresponding drugs were added to construct a network. Furthermore, the binding affinity between predicted drug candidates and ESCAassociated hub genes was calculated using molecular docking. Finally, 827 DEGs (|log2 foldchange|≥2; qvalue <0.05), which are principally involved in protein digestion and absorption (P<0.005), the plasminogenactivating cascade (P<0.01), as well as the 'biological regulation' of the Biological Process, 'membrane' of the Cellular Component and 'protein binding' of the Molecular Function categories, were obtained. Additionally, 11 hub genes were obtained from the PPI network (all degrees ≥30). Furthermore, the 15 first screen drugs were extracted from CMAP (score <0.85) and the 9 second screen drugs with 70 target genes were extracted from STITCH. Furthermore, another PPI analysis extracted 51 genes, and apigenin, baclofen, Prestwick685, menadione, butyl hydroxybenzoate, gliclazide and valproate were selected as drug candidates for ESCA. Those molecular docking results with a docking score of >5.52 indicated the significance of apigenin, Prestwick685 and menadione. The results of the present study may lead to novel drug candidates for ESCA, among which Prestwick685 and menadione were identified to be significant new drug candidates.
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Antineoplásicos/farmacología , Neoplasias Esofágicas/genética , Perfilación de la Expresión Génica/métodos , Redes Reguladoras de Genes , Antineoplásicos/química , Clofazimina/química , Clofazimina/farmacología , Bases de Datos Genéticas , Reposicionamiento de Medicamentos , Ensayos de Selección de Medicamentos Antitumorales , Neoplasias Esofágicas/tratamiento farmacológico , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Redes Reguladoras de Genes/efectos de los fármacos , Humanos , Modelos Moleculares , Simulación del Acoplamiento Molecular , Mapas de Interacción de Proteínas/efectos de los fármacos , Vitamina K 3/química , Vitamina K 3/farmacologíaRESUMEN
Xylella fastidiosa infects a wide range of plant hosts and causes Pierce's disease (PD) of grapevines. The type 1 multidrug resistance (MDR) efflux system is essential for pathogenicity and survival of bacterial pathogens in planta. X. fastidiosa, with a single MDR system, is significantly more vulnerable to inhibition by small-molecule treatments than most bacterial pathogens that typically carry redundant MDR systems. A high-throughput cell viability assay using a green fluorescent protein-marked strain of X. fastidiosa Temecula 1 was developed to screen two Prestwick combinatorial small-molecule libraries of drugs and phytochemicals (1,600 chemicals in total) approved by the Food and Drug Administration and European Medicines Agency for cell growth inhibition. The screens revealed 215 chemicals that inhibited bacterial growth by >50% at 50 µM concentrations. Seven chemicals proved to lyse X. fastidiosa cells at 25 µM, including four phytochemicals. Menadione (2-methyl-1,4-naphthoquinone, vitamin K) from the phytochemical library and benzethonium chloride (a topical disinfectant) from the chemical library both showed significant bactericidal activity against X. fastidiosa. Both menadione and benzethonium chloride foliar spray (15 and 5 mM, respectively) and soil drench (5 and 25 mM, respectively) treatments were equally effective in reducing PD symptoms by 54 to 59% and revealed that the effects of both chemical treatments became systemic. However, menadione was phytotoxic when applied as a foliar spray at effective concentrations, causing significant loss of photosynthetic capacity.
Asunto(s)
Bencetonio/química , Vitamina K 3/química , Vitis , Xylella , Enfermedades de las PlantasRESUMEN
Abnormal concentration of hydrogen peroxide (H2O2) in blood plasma and cells may lead to several diseases. Thus, it is important to develop a selective and sensitive method to monitor H2O2. In the present work, a novel nonenzymatic H2O2-sensing platform based on cobalt oxyhydroxide (CoOOH)/reduced graphene oxide (RGO) nanocomposite was fabricated. CoOOH nanoflakes were firstly synthesized via soft chemistry routes and then assembled on the surface of RGO. A series of characterizations by X-ray diffraction, X-ray photoelectron spectroscopy, and transmission electron microscopy demonstrated that hexagonal CoOOH nanoflakes were well distributed on the surface of RGO. The nanocomposite exhibited excellent electrochemical performance for H2O2 detection. Two linear ranges of 6-200 µM and 200-1500 µM were obtained, and the detection limit was 0.01 µM (signal-to-noise ratio was 3). The good performance was attributed to more exposed catalytic active sites of CoOOH nanoflakes compared with zero-dimensional nanoparticles and outstanding conductivity of RGO as well as their synergistic effect. Moreover, the nanocomposite was used to detect H2O2 from human serum and HeLa cells with satisfactory results. Graphical abstract á .