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1.
Biochem J ; 481(16): 1075-1096, 2024 Aug 21.
Artículo en Inglés | MEDLINE | ID: mdl-39105673

RESUMEN

Toxoplasma gondii is a widely distributed apicomplexan parasite causing toxoplasmosis, a critical health issue for immunocompromised individuals and for congenitally infected foetuses. Current treatment options are limited in number and associated with severe side effects. Thus, novel anti-toxoplasma agents need to be identified and developed. 1-Deoxy-d-xylulose 5-phosphate reductoisomerase (DXR) is considered the rate-limiting enzyme in the non-mevalonate pathway for the biosynthesis of the isoprenoid precursors isopentenyl pyrophosphate and dimethylallyl pyrophosphate in the parasite, and has been previously investigated for its key role as a novel drug target in some species, encompassing Plasmodia, Mycobacteria and Escherichia coli. In this study, we present the first crystal structure of T. gondii DXR (TgDXR) in a tertiary complex with the inhibitor fosmidomycin and the cofactor NADPH in dimeric conformation at 2.5 Šresolution revealing the inhibitor binding mode. In addition, we biologically characterize reverse α-phenyl-ß-thia and ß-oxa fosmidomycin analogues and show that some derivatives are strong inhibitors of TgDXR which also, in contrast with fosmidomycin, inhibit the growth of T. gondii in vitro. Here, ((3,4-dichlorophenyl)((2-(hydroxy(methyl)amino)-2-oxoethyl)thio)methyl)phosphonic acid was identified as the most potent anti T. gondii compound. These findings will enable the future design and development of more potent anti-toxoplasma DXR inhibitors.


Asunto(s)
Isomerasas Aldosa-Cetosa , Fosfomicina , Complejos Multienzimáticos , Toxoplasma , Toxoplasma/enzimología , Toxoplasma/efectos de los fármacos , Isomerasas Aldosa-Cetosa/antagonistas & inhibidores , Isomerasas Aldosa-Cetosa/química , Isomerasas Aldosa-Cetosa/metabolismo , Isomerasas Aldosa-Cetosa/genética , Fosfomicina/farmacología , Fosfomicina/análogos & derivados , Fosfomicina/química , Cristalografía por Rayos X , Complejos Multienzimáticos/antagonistas & inhibidores , Complejos Multienzimáticos/química , Complejos Multienzimáticos/metabolismo , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Proteínas Protozoarias/antagonistas & inhibidores , Proteínas Protozoarias/química , Proteínas Protozoarias/metabolismo , Proteínas Protozoarias/genética , NADP/metabolismo , NADP/química , Humanos , Modelos Moleculares , Oxidorreductasas/antagonistas & inhibidores , Oxidorreductasas/química , Oxidorreductasas/metabolismo
2.
Sci Rep ; 14(1): 12170, 2024 05 28.
Artículo en Inglés | MEDLINE | ID: mdl-38806590

RESUMEN

Tuberculosis (TB) continues to be a global health crisis, necessitating urgent interventions to address drug resistance and improve treatment efficacy. In this study, we validate lumazine synthase (RibH), a vital enzyme in the riboflavin biosynthetic pathway, as a potential drug target against Mycobacterium tuberculosis (M. tb) using a CRISPRi-based conditional gene knockdown strategy. We employ a high-throughput molecular docking approach to screen ~ 600,000 compounds targeting RibH. Through in vitro screening of 55 shortlisted compounds, we discover 3 compounds that exhibit potent antimycobacterial activity. These compounds also reduce intracellular burden of M. tb during macrophage infection and prevent the resuscitation of the nutrient-starved persister bacteria. Moreover, these three compounds enhance the bactericidal effect of first-line anti-TB drugs, isoniazid and rifampicin. Corroborating with the in silico predicted high docking scores along with favourable ADME and toxicity profiles, all three compounds demonstrate binding affinity towards purified lumazine synthase enzyme in vitro, in addition these compounds exhibit riboflavin displacement in an in vitro assay with purified lumazine synthase indicative of specificity of these compounds to the active site. Further, treatment of M. tb with these compounds indicate reduced production of flavin adenine dinucleotide (FAD), the ultimate end product of the riboflavin biosynthetic pathway suggesting the action of these drugs on riboflavin biosynthesis. These compounds also show acceptable safety profile in mammalian cells, with a high selective index. Hence, our study validates RibH as an important drug target against M. tb and identifies potent antimycobacterial agents.


Asunto(s)
Antituberculosos , Simulación del Acoplamiento Molecular , Mycobacterium tuberculosis , Mycobacterium tuberculosis/enzimología , Mycobacterium tuberculosis/efectos de los fármacos , Antituberculosos/farmacología , Antituberculosos/química , Complejos Multienzimáticos/antagonistas & inhibidores , Complejos Multienzimáticos/metabolismo , Descubrimiento de Drogas , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/genética , Proteínas Bacterianas/química , Humanos , Tuberculosis/tratamiento farmacológico , Tuberculosis/microbiología , Pruebas de Sensibilidad Microbiana , Animales
3.
Molecules ; 29(9)2024 Apr 23.
Artículo en Inglés | MEDLINE | ID: mdl-38731401

RESUMEN

The burden of human schistosomiasis, a known but neglected tropical disease in Sub-Saharan Africa, has been worrisome in recent years. It is becoming increasingly difficult to tackle schistosomiasis with praziquantel, a drug known to be effective against all Schistosoma species, due to reports of reduced efficacy and resistance. Therefore, this study seeks to investigate the antischistosomal potential of phytochemicals from Azadirachta indica against proteins that have been implicated as druggable targets for the treatment of schistosomiasis using computational techniques. In this study, sixty-three (63) previously isolated and characterized phytochemicals from A. indica were identified from the literature and retrieved from the PubChem database. In silico screening was conducted to assess the inhibitory potential of these phytochemicals against three receptors (Schistosoma mansoni Thioredoxin glutathione reductase, dihydroorotate dehydrogenase, and Arginase) that may serve as therapeutic targets for schistosomiasis treatment. Molecular docking, ADMET prediction, ligand interaction, MMGBSA, and molecular dynamics simulation of the hit compounds were conducted using the Schrodinger molecular drug discovery suite. The results show that Andrographolide possesses a satisfactory pharmacokinetic profile, does not violate the Lipinski rule of five, binds with favourable affinity with the receptors, and interacts with key amino acids at the active site. Importantly, its interaction with dihydroorotate dehydrogenase, an enzyme responsible for the catalysis of the de novo pyrimidine nucleotide biosynthetic pathway rate-limiting step, shows a glide score and MMGBSA of -10.19 and -45.75 Kcal/mol, respectively. In addition, the MD simulation shows its stability at the active site of the receptor. Overall, this study revealed that Andrographolide from Azadirachta indica could serve as a potential lead compound for the development of an anti-schistosomal drug.


Asunto(s)
Azadirachta , Dihidroorotato Deshidrogenasa , Simulación del Acoplamiento Molecular , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH , Esquistosomiasis , Azadirachta/química , Animales , Esquistosomiasis/tratamiento farmacológico , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/antagonistas & inhibidores , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/metabolismo , Humanos , Fitoquímicos/farmacología , Fitoquímicos/química , Simulación de Dinámica Molecular , Schistosoma mansoni/efectos de los fármacos , Schistosoma mansoni/enzimología , NADH NADPH Oxidorreductasas/antagonistas & inhibidores , NADH NADPH Oxidorreductasas/metabolismo , Extractos Vegetales/química , Extractos Vegetales/farmacología , Simulación por Computador , Esquistosomicidas/farmacología , Esquistosomicidas/química , Esquistosomicidas/uso terapéutico , Complejos Multienzimáticos/antagonistas & inhibidores , Complejos Multienzimáticos/metabolismo , Praziquantel/farmacología , Praziquantel/química , Praziquantel/uso terapéutico
4.
ACS Infect Dis ; 10(5): 1739-1752, 2024 05 10.
Artículo en Inglés | MEDLINE | ID: mdl-38647213

RESUMEN

Reverse analogs of the phosphonohydroxamic acid antibiotic fosmidomycin are potent inhibitors of the nonmevalonate isoprenoid biosynthesis enzyme 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR, IspC) of Plasmodium falciparum. Some novel analogs with large phenylalkyl substituents at the hydroxamic acid nitrogen exhibit nanomolar PfDXR inhibition and potent in vitro growth inhibition of P. falciparum parasites coupled with good parasite selectivity. X-ray crystallographic studies demonstrated that the N-phenylpropyl substituent of the newly developed lead compound 13e is accommodated in a subpocket within the DXR catalytic domain but does not reach the NADPH binding pocket of the N-terminal domain. As shown for reverse carba and thia analogs, PfDXR selectively binds the S-enantiomer of the new lead compound. In addition, some representatives of the novel inhibitor subclass are nanomolar Escherichia coli DXR inhibitors, whereas the inhibition of Mycobacterium tuberculosis DXR is considerably weaker.


Asunto(s)
Isomerasas Aldosa-Cetosa , Antimaláricos , Fosfomicina , Ácidos Hidroxámicos , Complejos Multienzimáticos , Plasmodium falciparum , Fosfomicina/farmacología , Fosfomicina/análogos & derivados , Fosfomicina/química , Isomerasas Aldosa-Cetosa/antagonistas & inhibidores , Isomerasas Aldosa-Cetosa/metabolismo , Isomerasas Aldosa-Cetosa/química , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/enzimología , Ácidos Hidroxámicos/farmacología , Ácidos Hidroxámicos/química , Antimaláricos/farmacología , Antimaláricos/química , Complejos Multienzimáticos/antagonistas & inhibidores , Complejos Multienzimáticos/metabolismo , Complejos Multienzimáticos/química , Cristalografía por Rayos X , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/síntesis química , Relación Estructura-Actividad , Escherichia coli/efectos de los fármacos , Escherichia coli/genética , Escherichia coli/enzimología , Modelos Moleculares , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/enzimología , Dominio Catalítico , Oxidorreductasas/antagonistas & inhibidores , Oxidorreductasas/metabolismo
5.
Chem Biol Interact ; 368: 110243, 2022 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-36374910

RESUMEN

Many environmental pollutants act as endocrine-disrupting compounds by inhibiting human placental 3ß-hydroxysteroid dehydrogenase/Δ5-4 isomerase type 1 (HSD3B1) and aromatase (CYP19A1) activities. In this study, we screened 13 chemicals of environmental concern for their ability to inhibit human HSD3B1 and CYP19A1 by measuring the conversion of pregnenolone to progesterone for HSD3B1 activity and the conversion of testosterone to 17ß-estradiol for CYP19A1 activity in human JEG-3 choriocarcinoma cell microsomes. HSD3B1 had an apparent Km of 0.323 µM and an apparent Vmax of 0.111 nmol/mg/min and CYP19A1 had an apparent Km of 56 nM and an apparent Vmax of 0.177 nmol/mg protein/min. 17ß-Estradiol, bisphenol A, and bisphenol AF competitively inhibited HSD3B1 with Ki values of 0.8, 284.1, and 141.2 µM, respectively, while diethylstilbestrol had a mixed inhibition on human HSD3B1 with the Ki of 8.0 µM. Ketoconazole, bisphenol A, and bisphenol AF noncompetitively inhibited CYP19A1 with Ki values of 10.3, 54.4, and 45.7 µM, respectively, while diethylstilbestrol and zearalenone competitively suppressed CYP19A1 with Ki values of 63.0 and 16.6 µM, respectively. Docking analysis showed that 17ß-estradiol, diethylstilbestrol, bisphenol A, and bisphenol AF bound the steroid binding pocket facing the catalytic residues Y155 and K159 of HSD3B1, and that ketoconazole, bisphenol A, and bisphenol AF bound heme binding pocket while diethylstilbestrol and zearalenone bound the steroid binding site of CYP19A1. In conclusion, 17ß-estradiol, diethylstilbestrol, bisphenol A, and bisphenol AF are human HSD3B1 inhibitors, and ketoconazole, zearalenone, diethylstilbestrol, bisphenol A, and bisphenol AF are human CYP19A1 inhibitors.


Asunto(s)
Inhibidores de la Aromatasa , Contaminantes Ambientales , Complejos Multienzimáticos , Femenino , Humanos , Embarazo , Aromatasa/metabolismo , Inhibidores de la Aromatasa/química , Inhibidores de la Aromatasa/farmacología , Línea Celular Tumoral , Dietilestilbestrol/toxicidad , Estradiol/metabolismo , Cetoconazol/toxicidad , Complejos Multienzimáticos/antagonistas & inhibidores , Zearalenona/toxicidad , Esteroide Isomerasas/antagonistas & inhibidores , Progesterona Reductasa/antagonistas & inhibidores , Fenoles/toxicidad , Contaminantes Ambientales/toxicidad
6.
Biochem Pharmacol ; 197: 114932, 2022 03.
Artículo en Inglés | MEDLINE | ID: mdl-35085541

RESUMEN

Neck pain and low back pain are two of the major diseases, which causes patients a low quantify of life and a heavy economic burden, intervertebral disc degeneration (IDD) contributes to them, and the mechanism is not totally clear. The increased inflammatory cytokines including interleukin (IL)-1ß and tumor necrosis factor (TNF)α and downstream signaling pathways are involved. Inositol requiring enzyme 1 (IRE1) is a crucial enzyme that regulates endoplasmic reticulum (ER) stress. It is reported that IRE1 plays an important role in the activation of NF-κB, PI3K/Akt and MAPK signaling pathways. Considering this, we performed a series of experiments in vitro and in vivo to evaluate the role of IRE1 in the progress of IDD. We demonstrated that IRE1 pathway was induced by IL-1ß, inhibition of IRE1 suppressed the matrix degeneration of NP cells and ameliorated IDD grade in the punctured rat model. Further results indicated that inhibition of IRE1 suppressed H2O2 induced cell senescence, IL-1ß-induced cellular reactive oxygen species (ROS) level and the activation of NF-κB, PI3K/Akt and MAPK signaling pathways. It also played a crucial role in the apoptosis of NP cells and the progress of macrophage polarization. Our findings demonstrated that inhibition of IRE1 could suppress the degeneration of NP cells and prevent IDD in vivo. IRE1 may be a potential target for IDD treatment.


Asunto(s)
Endorribonucleasas/metabolismo , Interleucina-1beta/metabolismo , Degeneración del Disco Intervertebral/metabolismo , Degeneración del Disco Intervertebral/prevención & control , Complejos Multienzimáticos/metabolismo , Núcleo Pulposo/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Células Cultivadas , Relación Dosis-Respuesta a Droga , Endorribonucleasas/antagonistas & inhibidores , Interleucina-1beta/antagonistas & inhibidores , Degeneración del Disco Intervertebral/patología , Masculino , Complejos Multienzimáticos/antagonistas & inhibidores , Núcleo Pulposo/patología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , ARN Interferente Pequeño/administración & dosificación , Ratas , Ratas Sprague-Dawley
7.
Nucleic Acids Res ; 50(3): 1484-1500, 2022 02 22.
Artículo en Inglés | MEDLINE | ID: mdl-35037045

RESUMEN

The SARS-CoV-2 coronavirus is the causal agent of the current global pandemic. SARS-CoV-2 belongs to an order, Nidovirales, with very large RNA genomes. It is proposed that the fidelity of coronavirus (CoV) genome replication is aided by an RNA nuclease complex, comprising the non-structural proteins 14 and 10 (nsp14-nsp10), an attractive target for antiviral inhibition. Our results validate reports that the SARS-CoV-2 nsp14-nsp10 complex has RNase activity. Detailed functional characterization reveals nsp14-nsp10 is a versatile nuclease capable of digesting a wide variety of RNA structures, including those with a blocked 3'-terminus. Consistent with a role in maintaining viral genome integrity during replication, we find that nsp14-nsp10 activity is enhanced by the viral RNA-dependent RNA polymerase complex (RdRp) consisting of nsp12-nsp7-nsp8 (nsp12-7-8) and demonstrate that this stimulation is mediated by nsp8. We propose that the role of nsp14-nsp10 in maintaining replication fidelity goes beyond classical proofreading by purging the nascent replicating RNA strand of a range of potentially replication-terminating aberrations. Using our developed assays, we identify drug and drug-like molecules that inhibit nsp14-nsp10, including the known SARS-CoV-2 major protease (Mpro) inhibitor ebselen and the HIV integrase inhibitor raltegravir, revealing the potential for multifunctional inhibitors in COVID-19 treatment.


Asunto(s)
Antivirales/farmacología , Evaluación Preclínica de Medicamentos , Exorribonucleasas/metabolismo , Genoma Viral/genética , Inestabilidad Genómica , SARS-CoV-2/enzimología , SARS-CoV-2/genética , Proteínas no Estructurales Virales/metabolismo , Proteínas Reguladoras y Accesorias Virales/metabolismo , ARN Polimerasa Dependiente de ARN de Coronavirus/metabolismo , Exorribonucleasas/antagonistas & inhibidores , Genoma Viral/efectos de los fármacos , Inestabilidad Genómica/efectos de los fármacos , Inestabilidad Genómica/genética , Inhibidores de Integrasa VIH/farmacología , Isoindoles/farmacología , Complejos Multienzimáticos/antagonistas & inhibidores , Complejos Multienzimáticos/metabolismo , Compuestos de Organoselenio/farmacología , ARN Viral/biosíntesis , ARN Viral/genética , Raltegravir Potásico/farmacología , SARS-CoV-2/efectos de los fármacos , Proteínas no Estructurales Virales/antagonistas & inhibidores , Proteínas Reguladoras y Accesorias Virales/antagonistas & inhibidores , Replicación Viral/efectos de los fármacos , Replicación Viral/genética
8.
Proc Natl Acad Sci U S A ; 118(39)2021 09 28.
Artículo en Inglés | MEDLINE | ID: mdl-34544865

RESUMEN

Bats are responsible for the zoonotic transmission of several major viral diseases, including those leading to the 2003 SARS outbreak and likely the ongoing COVID-19 pandemic. While comparative genomics studies have revealed characteristic adaptations of the bat innate immune system, functional genomic studies are urgently needed to provide a foundation for the molecular dissection of the viral tolerance in bats. Here we report the establishment of genome-wide RNA interference (RNAi) and CRISPR libraries for the screening of the model megabat, Pteropus alecto. We used the complementary RNAi and CRISPR libraries to interrogate P. alecto cells for infection with two different viruses: mumps virus and influenza A virus, respectively. Independent screening results converged on the endocytosis pathway and the protein secretory pathway as required for both viral infections. Additionally, we revealed a general dependence of the C1-tetrahydrofolate synthase gene, MTHFD1, for viral replication in bat cells and human cells. The MTHFD1 inhibitor, carolacton, potently blocked replication of several RNA viruses, including SARS-CoV-2. We also discovered that bats have lower expression levels of MTHFD1 than humans. Our studies provide a resource for systematic inquiry into the genetic underpinnings of bat biology and a potential target for developing broad-spectrum antiviral therapy.


Asunto(s)
Aminohidrolasas/genética , COVID-19/genética , Formiato-Tetrahidrofolato Ligasa/genética , Metilenotetrahidrofolato Deshidrogenasa (NADP)/genética , Complejos Multienzimáticos/genética , Pandemias , Aminohidrolasas/antagonistas & inhibidores , Animales , Antivirales/uso terapéutico , COVID-19/virología , Línea Celular , Quirópteros/genética , Quirópteros/virología , Formiato-Tetrahidrofolato Ligasa/antagonistas & inhibidores , Humanos , Metilenotetrahidrofolato Deshidrogenasa (NADP)/antagonistas & inhibidores , Antígenos de Histocompatibilidad Menor , Complejos Multienzimáticos/antagonistas & inhibidores , Virus ARN/genética , SARS-CoV-2/patogenicidad , Replicación Viral/genética , Tratamiento Farmacológico de COVID-19
9.
Invest New Drugs ; 39(6): 1493-1506, 2021 12.
Artículo en Inglés | MEDLINE | ID: mdl-34031786

RESUMEN

Background Human 3ß-hydroxysteroid dehydrogenase type 1 (HSD3B1) is an enzyme associated with steroidogenesis, however its' role in hepatocellular carcinoma (HCC) biology is unknown. Trilostane is an inhibitor of HSD3B1 and has been tested as a treatment for patients with breast cancer but has not been studied in patients with HCC. Methods and Results The expression of HSD3B1 in HCC tumors in 57 patients were examined. A total of 44 out of 57 tumors (77.2%) showed increased HSD3B1 expression. The increased HSD3B1 in tumors was significantly associated with advanced HCC. In vitro, the knockdown of HSD3B1 expression in Mahlavu HCC cells by a short hairpin RNA (shRNA) led to significant decreases in colony formation and cell migration. The suppression of clonogenicity in the HSD3B1-knockdown HCC cells was reversed by testosterone and 17ß-estradiol. Trilostane-mediated inhibition of HSD3B1 in different HCC cells also caused significant inhibition of clonogenicity and cell migration. In subcutaneous HCC Mahlavu xenografts, trilostane (30 or 60 mg/kg, intraperitoneal injection) significantly inhibited tumor growth in a dose-dependent manner. Furthermore, the combination of trilostane and sorafenib significantly enhanced the inhibition of clonogenicity and xenograft growth, surpassing the effects of each drug used alone, with no documented additional toxicity to animals. HSD3B1 blockade was found to suppress the phosphorylation of extracellular signal-regulated kinase (ERK). The decreased ERK phosphorylation was reversed by testosterone or 17b-estradiol. Conclusions Trilostane significantly inhibited the growth of HCC by inhibiting HSD3B1 function and augmenting the efficacy of sorafenib.


Asunto(s)
Carcinoma Hepatocelular/patología , Dihidrotestosterona/análogos & derivados , Neoplasias Hepáticas/patología , Complejos Multienzimáticos/antagonistas & inhibidores , Progesterona Reductasa/antagonistas & inhibidores , Sorafenib/farmacología , Esteroide Isomerasas/antagonistas & inhibidores , Anciano , Animales , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Dihidrotestosterona/administración & dosificación , Dihidrotestosterona/farmacología , Quimioterapia Combinada , Estradiol/farmacología , Femenino , Humanos , Masculino , Ratones , Ratones Desnudos , Persona de Mediana Edad , ARN Interferente Pequeño/efectos de los fármacos , Sorafenib/administración & dosificación , Testosterona/farmacología , Ensayos Antitumor por Modelo de Xenoinjerto
10.
Parasit Vectors ; 14(1): 225, 2021 Apr 26.
Artículo en Inglés | MEDLINE | ID: mdl-33902686

RESUMEN

BACKGROUND: Schistosomiasis is a chronic parasitic disease that affects millions of people's health worldwide. Because of the increasing drug resistance to praziquantel (PZQ), which is the primary drug for schistosomiasis, developing new drugs to treat schistosomiasis is crucial. Oxadiazole-2-oxides have been identified as potential anti-schistosomiasis reagents targeting thioredoxin glutathione reductase (TGR). METHODS: In this work, one of the oxadiazole-2-oxides derivatives furoxan was used as the lead compound to exploit a series of novel furoxan derivatives for studying inhibitory activity against both recombinant Schistosoma japonicum TGR containing selenium (rSjTGR-Sec) and soluble worm antigen protein (SWAP) containing wild-type Schistosoma japonicum TGR (wtSjTGR), in order to develop a new leading compound for schistosomiasis. Thirty-nine novel derivatives were prepared to test their activity toward both enzymes. The docking method was used to detect the binding site between the active molecule and SjTGR. The structure-activity relationship (SAR) of these novel furoxan derivatives was preliminarily analyzed. RESULTS: It was found that several new derivatives, including compounds 6a-6d, 9ab, 9bd and 9be, demonstrated greater activity toward rSjTGR-Sec or SWAP containing wtSjTGR than did furoxan. Interestingly, all intermediates bearing hydroxy (6a-6d) showed excellent inhibitory activity against both enzymes. In particular, compound 6d with trifluoromethyl on a pyridine ring was found to have much higher inhibition toward both rSjTGR-Sec (half-maximal inhibitory concentration, IC50,7.5nM) and SWAP containing wtSjTGR (IC50 55.8nM) than furoxan. Additionally, the docking method identified the possible matching sites between 6d and Schistosoma japonicum TGR (SjTGR), which theoretically lends support to the inhibitory activity of 6d. CONCLUSION: The data obtained herein showed that 6d with trifluoromethyl on a pyridine ring could be a valuable leading compound for further study.


Asunto(s)
Inhibidores Enzimáticos/farmacología , Complejos Multienzimáticos/antagonistas & inhibidores , NADH NADPH Oxidorreductasas/antagonistas & inhibidores , Oxadiazoles/farmacología , Schistosoma japonicum/efectos de los fármacos , Esquistosomiasis Japónica/tratamiento farmacológico , Animales , Antígenos Helmínticos/efectos de los fármacos , Cristalografía por Rayos X , Sistemas de Liberación de Medicamentos , Inhibidores Enzimáticos/uso terapéutico , Estructura Molecular , Oxadiazoles/química , Oxadiazoles/uso terapéutico , Schistosoma japonicum/enzimología , Selenio/química
11.
Carbohydr Polym ; 252: 117138, 2021 Jan 15.
Artículo en Inglés | MEDLINE | ID: mdl-33183597

RESUMEN

Bacterial adhesion infection caused by medical materials in clinical application has become a serious threat, and it urgently needs new strategies to deal with these clinical challenges. In this work, LED209, a highly selective histidine sensor kinase inhibitor of Gram-negative bacteria, was covalently attached on cellulose membrane (CM) via click reaction. The data of contact angle measurements, ATR-FTIR and X-ray photoelectron spectroscopy confirmed the successful synthesis of LED-CM. In addition, the results of antibacterial activity of the membranes shown that LED-CM exhibited excellent anti-adhesion ability to Enterohemorrhagic Escherichia coli (EHEC), and significantly reduced the formation of bacterial biofilm. Importantly, LED-CM was able to repress the expression of virulence genes in EHEC. Furthermore, LED209-functionalized cellulose membrane indicated no cytotoxicity to mammalian cells. Hence, our present work demonstrated that CM modified with LED209 possessed markedly anti-adhesion activity against EHEC, which offered a potent antimicrobial material for combating bacterial infections.


Asunto(s)
Proteínas de la Membrana Bacteriana Externa/antagonistas & inhibidores , Enzimas Inmovilizadas/farmacología , Infecciones por Escherichia coli/tratamiento farmacológico , Escherichia coli O157/efectos de los fármacos , Proteínas de Escherichia coli/antagonistas & inhibidores , Complejos Multienzimáticos/antagonistas & inhibidores , Sulfonamidas/farmacología , Animales , Antibacterianos/farmacología , Adhesión Bacteriana/efectos de los fármacos , Biopelículas/efectos de los fármacos , Celulosa/química , Membranas Artificiales , Ratones , Células 3T3 NIH
12.
PLoS Genet ; 16(11): e1009117, 2020 11.
Artículo en Inglés | MEDLINE | ID: mdl-33201894

RESUMEN

Glioblastoma is the most common and aggressive type of cancer in the brain; its poor prognosis is often marked by reoccurrence due to resistance to the chemotherapeutic agent temozolomide, which is triggered by an increase in the expression of DNA repair enzymes such as MGMT. The poor prognosis and limited therapeutic options led to studies targeted at understanding specific vulnerabilities of glioblastoma cells. Metabolic adaptations leading to increased synthesis of nucleotides by de novo biosynthesis pathways are emerging as key alterations driving glioblastoma growth. In this study, we show that enzymes necessary for the de novo biosynthesis of pyrimidines, DHODH and UMPS, are elevated in high grade gliomas and in glioblastoma cell lines. We demonstrate that DHODH's activity is necessary to maintain ribosomal DNA transcription (rDNA). Pharmacological inhibition of DHODH with the specific inhibitors brequinar or ML390 effectively depleted the pool of pyrimidines in glioblastoma cells grown in vitro and in vivo and impaired rDNA transcription, leading to nucleolar stress. Nucleolar stress was visualized by the aberrant redistribution of the transcription factor UBF and the nucleolar organizer nucleophosmin 1 (NPM1), as well as the stabilization of the transcription factor p53. Moreover, DHODH inhibition decreased the proliferation of glioblastoma cells, including temozolomide-resistant cells. Importantly, the addition of exogenous uridine, which reconstitutes the cellular pool of pyrimidine by the salvage pathway, to the culture media recovered the impaired rDNA transcription, nucleolar morphology, p53 levels, and proliferation of glioblastoma cells caused by the DHODH inhibitors. Our in vivo data indicate that while inhibition of DHODH caused a dramatic reduction in pyrimidines in tumor cells, it did not affect the overall pyrimidine levels in normal brain and liver tissues, suggesting that pyrimidine production by the salvage pathway may play an important role in maintaining these nucleotides in normal cells. Our study demonstrates that glioblastoma cells heavily rely on the de novo pyrimidine biosynthesis pathway to generate ribosomal RNA (rRNA) and thus, we identified an approach to inhibit ribosome production and consequently the proliferation of glioblastoma cells through the specific inhibition of the de novo pyrimidine biosynthesis pathway.


Asunto(s)
Antineoplásicos/farmacología , Neoplasias Encefálicas/tratamiento farmacológico , Nucléolo Celular/efectos de los fármacos , Glioblastoma/tratamiento farmacológico , Pirimidinas/biosíntesis , Animales , Antineoplásicos/uso terapéutico , Compuestos de Bifenilo/farmacología , Compuestos de Bifenilo/uso terapéutico , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Nucléolo Celular/metabolismo , Dihidroorotato Deshidrogenasa , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Glioblastoma/patología , Humanos , Ratones , Complejos Multienzimáticos/antagonistas & inhibidores , Complejos Multienzimáticos/metabolismo , Nucleofosmina , Orotato Fosforribosiltransferasa/antagonistas & inhibidores , Orotato Fosforribosiltransferasa/metabolismo , Orotidina-5'-Fosfato Descarboxilasa/antagonistas & inhibidores , Orotidina-5'-Fosfato Descarboxilasa/metabolismo , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/antagonistas & inhibidores , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/metabolismo , ARN Ribosómico/biosíntesis , Ribosomas/efectos de los fármacos , Ribosomas/metabolismo , Estrés Fisiológico/efectos de los fármacos , Transcripción Genética/efectos de los fármacos , Proteína p53 Supresora de Tumor/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto
13.
Acta Trop ; 210: 105621, 2020 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-32659283

RESUMEN

The carcinogenic liver fluke Opisthorchis viverrini causes several hepatobiliary diseases including a bile duct cancer-cholangiocarcinoma (CCA), which is a major public health problem in many countries in the Greater Mekong Sub-region. Praziquantel is the main drug against this parasite, however, reduced drug efficacy has been observed in some endemic areas. Therefore, alternative drugs are needed to prepare for praziquantel resistance in the future. The selenoprotein thioredoxin glutathione reductase (TGR) enzyme, which plays a crucial role in cellular redox balance of parasitic flatworms, has been shown as a potential drug target against these parasites. Hence, this study aimed to investigate the TGR of O. viverrini and assess its potential as a drug target. An open reading frame (ORF) that encodes O. viverrini TGR (Ov-TGR) was cloned from an O. viverrini cDNA library and the nucleotide were sequenced. The 1,812 nucleotides of the Ov-TGR full ORF encoded a polypeptide of 603 amino acid residues with a predicted molecular mass of 66 kDa. The putative amino acid sequence shared 55-96.8% similarities with TGRs from other helminths and mammals. Phylogenetic analysis revealed a close relationship of Ov-TGR with that of other trematodes. The ORF of Ov-TGR was inserted into pABC2 plasmid and transformed into Escherichia coli strain C321.ΔA to facilitate selenocysteine incorporation. The recombinant Ov-TGR (rOv-TGR-SEC) was expressed as a soluble protein and detected as a dimer form in the non-reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Its thioredoxin reductase (TrxR) and glutathione reductase (GR) activities were detected using DTNB, Trx and GSSG substrates with the Michaelis constant (Km) of 292.6 ± 52.3 µM, 8.09 ± 1.91 µM and 13.74 ± 1.2 µM, respectively. The TGR enzyme activities were effectively inhibited by a well-known inhibitor, auranofin in a dose-dependent manner. Moreover, auranofin expressed a lethal toxic effect on both newly excysted juveniles (NEJs) and adult worms of O. viverrini in vitro. Taken together, these results indicated that Ov-TGR is crucial for O. viverrini survival and maybe a potential target for the development of novel agents against opisthorschiasis.


Asunto(s)
Complejos Multienzimáticos/fisiología , NADH NADPH Oxidorreductasas/fisiología , Opisthorchis/enzimología , Animales , Auranofina/farmacología , Complejos Multienzimáticos/antagonistas & inhibidores , Complejos Multienzimáticos/genética , NADH NADPH Oxidorreductasas/antagonistas & inhibidores , NADH NADPH Oxidorreductasas/genética , Sistemas de Lectura Abierta , Opisthorchis/efectos de los fármacos , Filogenia
14.
J Neuroinflammation ; 17(1): 152, 2020 May 06.
Artículo en Inglés | MEDLINE | ID: mdl-32375838

RESUMEN

BACKGROUND: Inhibition of inositol-requiring enzyme-1 alpha (IRE1α), one of the sensor signaling proteins associated with endoplasmic reticulum (ER) stress, has been shown to alleviate brain injury and improve neurological behavior in a neonatal hypoxic-ischemic encephalopathy (HIE) rat model. However, there is no information about the role of IRE1α inhibitor as well as its molecular mechanisms in preventing neuronal pyroptosis induced by NLRP1 (NOD-, LRR- and pyrin domain-containing 1) inflammasome. In the present study, we hypothesized that IRE1α can degrade microRNA-125-b-2-3p (miR-125-b-2-3p) and activate NLRP1/caspased-1 pathway, and subsequently promote neuronal pyroptosis in HIE rat model. METHODS: Ten-day old unsexed rat pups were subjected to hypoxia-ischemia (HI) injury, and the inhibitor of IRE1α, STF083010, was administered intranasally at 1 h after HI induction. AntimiR-125 or NLRP1 activation CRISPR was administered by intracerebroventricular (i.c.v) injection at 24 h before HI induction. Immunofluorescence staining, western blot analysis, reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR), brain infarct volume measurement, neurological function tests, and Fluoro-Jade C staining were performed. RESULTS: Endogenous phosphorylated IRE1α (p-IRE1α), NLRP1, cleaved caspase-1, interleukin-1ß (IL-1ß), and interleukin-18 (IL-18) were increased and miR-125-b-2-3p was decreased in HIE rat model. STF083010 administration significantly upregulated the expression of miR-125-b-2-3p, reduced the infarct volume, improved neurobehavioral outcomes and downregulated the protein expression of NLRP1, cleaved caspase-1, IL-1ß and IL-18. The protective effects of STF083010 were reversed by antimiR-125 or NLRP1 activation CRISPR. CONCLUSIONS: IRE1α inhibitor, STF083010, reduced neuronal pyroptosis at least in part via miR-125/NLRP1/caspase-1 signaling pathway after HI.


Asunto(s)
Endorribonucleasas/antagonistas & inhibidores , Hipoxia-Isquemia Encefálica/patología , MicroARNs/metabolismo , Complejos Multienzimáticos/antagonistas & inhibidores , Proteínas del Tejido Nervioso/metabolismo , Neuronas/patología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Animales , Animales Recién Nacidos , Modelos Animales de Enfermedad , Hipoxia-Isquemia Encefálica/metabolismo , Inflamasomas/efectos de los fármacos , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Piroptosis/efectos de los fármacos , Piroptosis/fisiología , Ratas , Ratas Sprague-Dawley , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Sulfonamidas/farmacología , Tiofenos/farmacología
15.
Acta Trop ; 207: 105488, 2020 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-32277926

RESUMEN

Toxoplasma gondii (T. gondii), an obligatory intracellular parasite, is the etiologic agent of toxoplasmosis. Dihydrofolate reductase-thymidylate synthase (DHFR-TS) is one of the most important enzymes in toxoplasma folic acid cycle. Due to the emergence of resistance in RH strain of T. gondii against pyrimethamine that acts via DHFR-TS inhibition and also the crucial role of small interference RNA (siRNA) technology in gene silencing, we aimed to use siRNA to knock down DHFR-TS gene expression in T. gondii as a therapeutic target against toxoplasmosis in a mouse model. Based on the DHFR-TS gene sequence, siRNA was designed. The siRNAs were transfected into the parasites by electroporation. Total RNA was extracted using RNX-Plus kit. The viability of parasite was assessed by methylthiazole tetrazolium (MTT). The survival time of mice challenged with siRNA-treated T.gondii were compared to the control group infected with the same amount of wild-type tachyzoites. The viability of siRNA-embedded parasites was 70.7% (29.3% decreased) compared to the wild-type parasite as control (P = 0.0001). The transcription level of siRNA-transfected parasites was reduced to 17.4% (82.6% inhibition) (P = 0.016). The in vivo assessment showed that the mean survival time of the mice inoculated with modified parasites was increased about 2 days after the death of all mice in the control group. The designed siRNAs in the current study were able to silence the DHFR-TS gene efficiently. This silencing led to a decrease in viability of the parasites and an increase in the survival time of the parasites-treated mice.


Asunto(s)
Complejos Multienzimáticos/antagonistas & inhibidores , ARN Interferente Pequeño/genética , Timidilato Sintasa/antagonistas & inhibidores , Toxoplasma/enzimología , Toxoplasmosis/terapia , Animales , Ratones , Complejos Multienzimáticos/genética , Pirimetamina/farmacología , Tetrahidrofolato Deshidrogenasa/genética , Timidilato Sintasa/genética , Toxoplasma/efectos de los fármacos
16.
ACS Infect Dis ; 6(5): 893-895, 2020 05 08.
Artículo en Inglés | MEDLINE | ID: mdl-32159329

RESUMEN

Species of the blood fluke Schistosoma are responsible for schistosomiasis, the second most common parasitic disease, which is prevalent particularly in poor communities. Under redox pressure, schistosomes survive in mammalian hosts with the help of thioredoxin glutathione reductase, which is an essential selenoenzyme. A recent study identified compounds with extremely potent antischistosome activity. Most importantly, certain compounds were active against all major schistosomes across different life cycle stages, where even praziquantel, the drug of choice, fails. The data offer compounds that exceed WHO standards for leads for schistosomiasis therapy activity. The work may serve as the basis for the development of new antischistosome compounds.


Asunto(s)
Complejos Multienzimáticos/antagonistas & inhibidores , NADH NADPH Oxidorreductasas/antagonistas & inhibidores , Schistosoma/efectos de los fármacos , Esquistosomicidas/farmacología , Animales
17.
Biosci Biotechnol Biochem ; 84(5): 1023-1029, 2020 May.
Artículo en Inglés | MEDLINE | ID: mdl-31942834

RESUMEN

Indoxyl sulfate (IS), a uremic toxin, is a sulfate-conjugated metabolite originated from tryptophan. Accumulating uremic toxins may worsen renal diseases and further complicate related disorders including impaired immune functions under oxidative stress conditions. However, it has remained unclear whether or not IS can directly cause the cellular immune dysfunction. We investigated the effects of IS on the intracellular oxidation level and phagocytic activity in a HL-60-differantiated human macrophage cell model. Incubation of the cells in the presence of IS resulted in increasing intracellular oxidation level and decreasing phagocytic activity. In addition to inhibitors for NADH oxidase (NOX), organic anion transporting polypeptide2B1 (OATP2B1), protein kinase C (PKC), and phosphoinositide 3-kinase (PI3K), a representative antioxidant Trolox, was also shown to significantly relieve the IS-induced oxidation and restore weakened phagocytosis. Collectively, IS may directly down-regulate the phagocytic immune function of macrophages through the oxidation mechanisms including OATP2B1, PKC, PI3K, and NOX pathways. Abbreviations: CKD: Chronic kidney disease; IS: Indoxyl sulfate; ROS: Reactive oxygen species; NOX: NADH oxidase; OATP2B1: Organic anion transporting polypeptide2B1; PKC: Protein kinase C; PI3K: Phosphoinositide 3-kinase; 2-APT: 2-acetylphenothiazine.


Asunto(s)
Diferenciación Celular/efectos de los fármacos , Indicán/farmacología , Espacio Intracelular/metabolismo , Macrófagos/efectos de los fármacos , Fagocitosis/efectos de los fármacos , Toxinas Biológicas/farmacología , Antioxidantes/farmacología , Cromanos/farmacología , Células HL-60 , Humanos , Macrófagos/metabolismo , Complejos Multienzimáticos/antagonistas & inhibidores , Complejos Multienzimáticos/metabolismo , NADH NADPH Oxidorreductasas/antagonistas & inhibidores , NADH NADPH Oxidorreductasas/metabolismo , Transportadores de Anión Orgánico/antagonistas & inhibidores , Transportadores de Anión Orgánico/metabolismo , Oxidación-Reducción , Estrés Oxidativo/efectos de los fármacos , Fagocitosis/inmunología , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3/farmacología , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/efectos de los fármacos
18.
ACS Infect Dis ; 6(3): 393-405, 2020 03 13.
Artículo en Inglés | MEDLINE | ID: mdl-31939288

RESUMEN

Schistosomiasis is a widespread human parasitic disease currently affecting over 200 million people. Chemotherapy for schistosomiasis relies exclusively on praziquantel. Although significant advances have been made in recent years to reduce the incidence and intensity of schistosome infections, these gains will be at risk should drug-resistant parasites evolve. Thioredoxin glutathione reductase (TGR) is a selenoprotein of the parasite essential for the survival of schistosomes in the mammalian host. Several high-throughput screening campaigns have identified inhibitors of Schistosoma mansoni TGR. Follow up analyses of select active compounds form the basis of the present study. We identified eight compounds effective against ex vivo worms. Compounds 1-5 are active against all major species and development stages. The ability of these compounds to target immature worms is especially critical because praziquantel is poorly active against this stage. Compounds 1-5, 7, and 8 displayed schistosomicidal activity even after only 1 h incubation with the worms. Compounds 1-4 meet or exceed standards set by the World Health Organization for leads for schistosomiasis therapy activity. The mechanism of TGR inhibition was studied further with wild-type and mutant TGR proteins. Compounds 4-6 were found to induce an nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity in TGR, leading to the production of superoxide and hydrogen peroxide. Collectively, this effort has identified several active compound series that may serve as the basis for the development of new schistosomicidal compounds.


Asunto(s)
Inhibidores Enzimáticos/farmacología , Complejos Multienzimáticos/antagonistas & inhibidores , NADH NADPH Oxidorreductasas/antagonistas & inhibidores , Schistosoma mansoni/efectos de los fármacos , Schistosoma mansoni/enzimología , Esquistosomiasis/tratamiento farmacológico , Esquistosomicidas/farmacología , Animales , Descubrimiento de Drogas , Ensayos Analíticos de Alto Rendimiento , Concentración 50 Inhibidora , Ratones , Complejos Multienzimáticos/genética , NADH NADPH Oxidorreductasas/genética , NADP/metabolismo , Oxidación-Reducción/efectos de los fármacos
19.
Int J Biol Macromol ; 157: 626-640, 2020 Aug 15.
Artículo en Inglés | MEDLINE | ID: mdl-31786301

RESUMEN

Acetylation of proteins is vital and mediate many processes within the cells like protein interactions, intercellular localization, protein stability, transcriptional regulation, enzyme activity and many more. Acetylation, an evolutionarily conserved process, attracted more attention due to its key regulatory role in many cellular processes and its effect on proteome and metabolome. In eukaryotes, protein acetylation also contribute to the epigenetic regulation of gene expression. Acetylation involves the transfer of acetyl group from donor acetyl coenzyme A to a suitable acceptor molecule and the reaction is catalyzed by acetyltransferase enzymes. The review focuses on current understanding of different acetyltransferase families: their discovery, structure and catalytic mechanism in fungal species. Fungal acetyltransferases use divergent catalytic mechanisms and carry out catalysis in a substrate-specific manner. The studies have explored different fungal acetyltransferases in relation to secondary metabolite production and the fungal pathogenesis. Although, the functions and catalytic mechanism of acetyltransferases are well known, however further enhanced knowledge may improve their utilization in various applications of biotechnology.


Asunto(s)
Acetiltransferasas/química , Acetiltransferasas/metabolismo , Antifúngicos/química , Inhibidores Enzimáticos/química , Proteínas Fúngicas/química , Hongos/enzimología , Modelos Moleculares , Conformación Molecular , Acetiltransferasas/antagonistas & inhibidores , Antifúngicos/farmacología , Catálisis , Descubrimiento de Drogas , Inhibidores Enzimáticos/farmacología , Proteínas Fúngicas/antagonistas & inhibidores , Proteínas Fúngicas/metabolismo , Humanos , Complejos Multienzimáticos/antagonistas & inhibidores , Complejos Multienzimáticos/química , Complejos Multienzimáticos/metabolismo , Relación Estructura-Actividad , Especificidad por Sustrato
20.
Chemosphere ; 245: 125597, 2020 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-31864041

RESUMEN

Acephate is an organophosphate pesticide. It is widely used. However, whether it inhibits androgen synthesis and metabolism remains unclear. In the current study, we investigated the effect of acephate on the inhibition of androgen synthetic and metabolic pathways in rat immature Leydig cells after 3-h culture. Acephate inhibited basal androgen output in a dose-dependent manner with the inhibition starting at 0.5 µM. It significantly inhibited luteinizing hormone and 8-Br-cAMP stimulated androgen output at 50 µM. It significantly inhibited progesterone-mediated androgen output at 50 µM. Further study demonstrated that acephate down-regulated the expression of Hsd3b1 and its protein at ≥ 0.5 µM, Lhcgr at 5 µM and Star at 50 µM. Acephate directly blocked rat testicular HSD3B1 activity at 50 µM. Acephate did not affect other androgen synthetic and metabolic enzyme activities as well as ROS production, proliferation, and apoptosis of immature Leydig cells. In conclusion, acephate targets LHCGR, STAR, and HSD3B1, thus blocking androgen synthesis in rat immature Leydig cells and HSD3B1 is being the most sensitive target of acephate.


Asunto(s)
Andrógenos/metabolismo , Células Intersticiales del Testículo/efectos de los fármacos , Compuestos Organotiofosforados/farmacología , Fosforamidas/farmacología , 8-Bromo Monofosfato de Adenosina Cíclica/análogos & derivados , 8-Bromo Monofosfato de Adenosina Cíclica/antagonistas & inhibidores , Animales , Apoptosis/efectos de los fármacos , Células Cultivadas , Células Intersticiales del Testículo/metabolismo , Hormona Luteinizante/antagonistas & inhibidores , Hormona Luteinizante/metabolismo , Masculino , Complejos Multienzimáticos/antagonistas & inhibidores , Progesterona/farmacología , Progesterona Reductasa/antagonistas & inhibidores , Ratas , Ratas Sprague-Dawley , Receptores de HL/antagonistas & inhibidores , Esteroide Isomerasas/antagonistas & inhibidores , Testículo/efectos de los fármacos , Testículo/metabolismo
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