RESUMO
Structural motifs containing nitrogen-nitrogen (N-N) bonds are prevalent in a large number of clinical drugs and bioactive natural products. Hydrazine (N2H4) serves as a widely utilized building block for the preparation of these N-N-containing molecules in organic synthesis. Despite its common use in chemical processes, no enzyme has been identified to catalyze the incorporation of free hydrazine in natural product biosynthesis. Here, we report that a hydrazine transferase catalyzes the condensation of N2H4 and an aromatic polyketide pathway intermediate, leading to the formation of a rare N-aminolactam pharmacophore in the biosynthesis of broad-spectrum antibiotic albofungin. These results expand the current knowledge on the biosynthetic mechanism for natural products with N-N units and should facilitate future development of biocatalysts for the production of N-N-containing chemicals.
Assuntos
Hidrazinas , Hidrazinas/química , Hidrazinas/metabolismo , Antibacterianos/química , Antibacterianos/biossíntese , Antibacterianos/farmacologia , Streptomyces/enzimologia , Streptomyces/metabolismo , Lactamas/química , Lactamas/metabolismo , FarmacóforoRESUMO
The role of hydrazine (N2H4) in anammox metabolism has been widely studied; however, studies on N2H4 biosynthesis by anammox bacteria are limited in the literature. In this context, the current research aims to investigate the enhancement of biological N2H4 production in the anammox process in a long-term manner. The experimental studies started with the optimization of the operating conditions to achieve maximum N2H4 accumulation. Under favorable conditions (pH = 8.97 ± 0.08; T = 35.5 ± 0.5 °C; initial hydroxylamine dose = 1.46 ± 0.01 mM), 17.16 ± 0.64 mg L-1 of N2H4 accumulated in the batch systems. The continuity of N2H4 bioproduction was then evaluated by long-term observations. A continuous flow bioreactor was operated in four consecutive manipulated periods under optimized conditions. In the long-term operated bioreactor, 55.10 ± 0.30 mg L-1 N2H4 was accumulated at optimal conditions, which was 2.5 times higher than reported in the literature. Although manipulation of the bioreactor operating conditions initially resulted in a significant increase in N2H4 bioaccumulation, it subsequently caused a severe deterioration in anammox activity. However, this could be mitigated by increasing the biomass concentration in the anammox systems. In addition, the relative abundance of Candidatus Kuenenia decreased by 1.88% throughout the long-term operation.
Assuntos
Reatores Biológicos , Hidrazinas , Reatores Biológicos/microbiologia , Hidrazinas/metabolismo , Anaerobiose , Bactérias/metabolismo , Oxirredução , BiomassaRESUMO
Nitrogen-Nitrogen (N-N) bond-containing functional groups in natural products and synthetic drugs play significant roles in exerting biological activities. The mechanisms of N-N bond formation in natural organic molecules have garnered increasing attention over the decades. Recent advances have illuminated various enzymatic and nonenzymatic strategies, and our understanding of natural N-N bond construction is rapidly expanding. A group of didomain proteins with zinc-binding cupin/methionyl-tRNA synthetase (MetRS)-like domains, also known as hydrazine synthetases, generates amino acid-based hydrazines, which serve as key biosynthetic precursors of diverse N-N bond-containing functionalities such as hydrazone, diazo, triazene, pyrazole, and pyridazinone groups. In this review, we summarize the current knowledge on hydrazine synthetase mechanisms and the various pathways employing this unique bond-forming machinery.
Assuntos
Hidrazinas , Hidrazinas/química , Hidrazinas/metabolismo , Metionina tRNA Ligase/metabolismo , Bactérias/enzimologia , Bactérias/metabolismo , Vias BiossintéticasRESUMO
Acetylhydrazine (AcHZ), a major human metabolite of the widely-used anti-tuberculosis drug isoniazid (INH), was considered to be responsible for its serious hepatotoxicity and potentially fatal liver injury. It has been proposed that reactive radical species produced from further metabolic activation of AcHZ might be responsible for its hepatotoxicity. However, the exact nature of such radical species remains not clear. Through complementary applications of ESR spin-trapping and HPLC/MS methods, here we show that the initial N-centered radical intermediate can be detected and identified from AcHZ activated by transition metal ions (Mn(III)Acetate and Mn(III) pyrophosphate) and myeloperoxidase. The exact location of the radical was found to be at the distal-nitrogen of the hydrazine group by 15N-isotope-labeling techniques via using 15N-labeled AcHZ we synthesized. Additionally, the secondary C-centered radical was identified unequivocally as the reactive acetyl radical by complementary applications of ESR spin-trapping and persistent radical TEMPO trapping coupled with HPLC/MS analysis. This study represents the first detection and unequivocal identification of the initial N-centered radical and its exact location, as well as the reactive secondary acetyl radical. These findings should provide new perspectives on the molecular mechanism of AcHZ activation, which may have potential biomedical and toxicological significance for future research on the mechanism of INH-induced hepatotoxicity.
Assuntos
Doença Hepática Induzida por Substâncias e Drogas , Hidrazinas , Humanos , Hidrazinas/metabolismo , Isoniazida/metabolismo , Antituberculosos/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica , Radicais LivresRESUMO
Anaerobic ammon ium oxidizing (anammox) bacteria oxidize ammonium and reduce nitrite, producing N2, and could play a major role in energy-optimized wastewater treatment. However, sensitivity to various environmental conditions and slow growth currently hinder their wide application. Here, we attempted to determine online the effect of environmental stresses on anammox bacteria by using an overnight batch activity test with whole cells, in which anammox activity was calculated by quantifying N2 production via headspace-pressure monitoring. A planktonic mixed culture dominated by "Candidatus Kuenenia stuttgartiensis" strain CSTR1 was cultivated in a 30-L semi-continuous stirring tank reactor. In overnight resting-cell anammox activity tests, oxygen caused strong inhibition of anammox activity, which was reversed by sodium sulfite (30 µM). The tested antibiotics sulfamethoxazole, kanamycin, and ciprofloxacin elicited their effect on a dose-dependent manner; however, strain CSTR1 was highly resistant to sulfamethoxazole. Anammox activity was improved by activated carbon and Fe2O3. Protein expression analysis from resting cells after anammox activity stimulation revealed that NapC/NirT family cytochrome c (KsCSTR_12840), hydrazine synthase, hydrazine dehydrogenase, hydroxylamine oxidase, and nitrate:nitrite oxidoreductase were upregulated, while a putative hydroxylamine oxidoreductase HAO (KsCSTR_49490) was downregulated. These findings contribute to the growing knowledge on anammox bacteria physiology, eventually leading to the control of anammox bacteria growth and activity in real-world application. KEY POINTS: ⢠Sulfite additions can reverse oxygen inhibition of the anammox process ⢠Anammox activity was improved by activated carbon and ferric oxide ⢠Sulfamethoxazole marginally affected anammox activity.
Assuntos
Compostos de Amônio , Nitritos , Oxirredução , Nitritos/metabolismo , Carvão Vegetal , Compostos de Amônio/metabolismo , Bactérias/metabolismo , Antibacterianos/metabolismo , Hidrazinas/metabolismo , Sulfametoxazol/metabolismo , Anaerobiose , Reatores BiológicosRESUMO
As a common reactive metabolite in living organisms, abnormal levels of formaldehyde may cause diseases such as cancer and Alzheimer's disease. Therefore, it is important to develop a sensitive and efficient method to understand the role of formaldehyde in physiology and pathology. Herein, a new fluorescent probe 4-phenyl-2-(trifluoromethyl) quinolin-7-hydrazino (QH-FA) was prepared for the detection of formaldehyde in near-total aqueous media with hydrazine as the reaction site and quinoline derivatives as the fluorophore. After reacting with formaldehyde, the hydrazine group formed methylenehydrazine and the fluorescence was significantly enhanced (223-fold) with large Stokes shift of 140 nm. Furthermore, the response of QH-FA to formaldehyde could be finished with in only 10 min with good selectivity, and can distinguish formaldehyde from other aldehydes. More remarkably, the estimated limit of detection of QH-FA is 8.1 nM, which is superior to those of previously reported formaldehyde fluorescent probes. At the end, we detected formaldehyde in cells and zebrafish using QH-FA in a near-total aqueous system and obtained fluorescence images by confocal microscopy.
Assuntos
Quinolinas , Peixe-Zebra , Animais , Humanos , Hidrazinas/metabolismo , Células HeLa , Formaldeído , Corantes Fluorescentes/metabolismoRESUMO
Chromosomal region maintenance 1 (CRM1 also known as Xpo1 and exportin-1) is the receptor for the nuclear export controlling the intracellular localization and function of many cellular and viral proteins that play a crucial role in viral infections and cancer. The inhibition of CRM1 has emerged as a promising therapeutic approach to interfere with the lifecycle of many viruses, for the treatment of cancer, and to overcome therapy resistance. Recently, selinexor has been approved as the first CRM1 inhibitor for the treatment of multiple myeloma, providing proof of concept for this therapeutic option with a new mode of action. However, selinexor is associated with dose-limiting toxicity and hence, the discovery of alternative small molecule leads that could be developed as less toxic anticancer and antiviral therapeutics will have a significant impact in the clinic. Here, we report a CRM1 inhibitor discovery platform. The development of this platform includes reporter cell lines that monitor CRM1 activity by using red fluorescent protein or green fluorescent protein-labeled HIV-1 Rev protein with a strong heterologous nuclear export signal. Simultaneously, the intracellular localization of other proteins, to be interrogated for their capacity to undergo CRM1-mediated export, can be followed by co-culturing stable cell lines expressing fluorescent fusion proteins. We used this platform to interrogate the mode of nuclear export of several proteins, including PDK1, p110α, STAT5A, FOXO1, 3, 4 and TRIB2, and to screen a compound collection. We show that while p110α partially relies on CRM1-dependent nuclear export, TRIB2 is exported from the nucleus in a CRM1-independent manner. Compound screening revealed the striking activity of an organoselenium compound on the CRM1 nuclear export receptor.
Assuntos
HIV-1 , Transporte Ativo do Núcleo Celular , HIV-1/metabolismo , Carioferinas/metabolismo , Triazóis/metabolismo , Hidrazinas/farmacologia , Hidrazinas/metabolismo , Núcleo Celular/metabolismoRESUMO
Bifenazate is a novel acaricide that has been widely used to control spider mites. Interestingly, we found bifenazate had a biological activity against the diamondback moth (Plutella xylostella), one of the most economically important pests on crucifer crops around the world. However, the molecular mechanisms underlying the response of P. xylostella to bifenazate treatment are not clear. In this study, we first estimated the LC30 dose of bifenazate for third-instar P. xylostella larvae. Then, in order to identify genes that respond to the treatment of this insecticide, the comparative transcriptome profiles were used to analyze the gene expression changes in P. xylostella larvae after exposure to LC30 of bifenazate. In total, 757 differentially expressed genes (DEGs) between bifenazate-treated and control P. xylostella larvae were identified, in which 526 and 231 genes were up-regulated and down-regulated, respectively. The further Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that the xenobiotics metabolisms pathway was significantly enriched, with ten detoxifying enzyme genes (four P450s, five glutathione S-transferases (GSTs), and one UDP-Glucuronosyltransferase (UGT)) were up-regulated, and their expression patterns were validated by qRT-PCR as well. Interestingly, the present results showed that 17 cuticular protein (CP) genes were also remarkably up-regulated, including 15 CPR family genes. Additionally, the oxidative phosphorylation pathway was found to be activated with eight mitochondrial genes up-regulated in bifenazate-treated larvae. In contrast, we found some genes that were involved in tyrosine metabolism and purine pathways were down-regulated, indicating these two pathways of bifenazate-exposed larvae were significantly inhibited. In conclusion, the present study would help us to better understand the molecular mechanisms of sublethal doses of bifenazate detoxification and action in P. xylostella.
Assuntos
Mariposas , Animais , Mariposas/genética , Mariposas/metabolismo , Perfilação da Expressão Gênica , Carbamatos , Hidrazinas/metabolismo , Transcriptoma , Larva/genéticaRESUMO
The environmental factors and genetic vulnerability trigger the inflammatory bowel diseases (IBDs) such as ulcerative colitis and Crohn's disease. Furthermore, the oxidative stress and inflammatory cytokines have been implicated in the aggravation of the IBDs. The aim of the present study was to investigate the effect of N-(benzylidene)-2-((2-hydroxynaphthalen-1-yl)diazenyl)benzohydrazides (NCHDH and NTHDH) compounds against the DSS-induced colitis in mice. The colitis was induced by 5% dextran sulfate sodium (DSS) dissolved in normal saline for 5 days. The effect of the NCHDH and NTHDH on the behavioral, biochemical, histological, and immunohistological parameters was assessed. The NCHDH and NTHDH treatment improved the behavioral parameters such as food intake, disease activity index, and diarrhea score significantly compared to DSS control. The NCHDH and NTHDH treatments significantly increased the antioxidant enzymes, whereas oxidative stress markers were markedly reduced. Similarly, the NCHDH and NTHDH treatments significantly suppressed the activity of nitric oxide (NO), myeloperoxidase (MPO), and eosinophil peroxidase (EPO). The histological studies showed a significant reduction in inflammation, immune cell infiltration, and fibrosis in the NCHDH- and NTHDH-treated groups. The immunohistochemical results demonstrated that NCHDH and NTHDH treatments markedly increase the expression level of Nrf2, HO-1 (hemeoxygenase-1), TRX (thioredoxin reductase), and IκB compared to the DSS-induced group. In the same way, the NCHDH and NTHDH significantly reduced the NF-κB and COX-2 (cyclooxygenase-2) expression levels. The NCHDH and NTHDH treatment significantly improved the symptoms associated with colitis via inducing antioxidants and attenuating oxidative stress markers.
Assuntos
Colite , NF-kappa B , Animais , Antioxidantes/efeitos adversos , Colite/induzido quimicamente , Colite/tratamento farmacológico , Colite/metabolismo , Colo , Sulfato de Dextrana/efeitos adversos , Sulfato de Dextrana/metabolismo , Modelos Animais de Doenças , Hidrazinas/efeitos adversos , Hidrazinas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Fator 2 Relacionado a NF-E2/metabolismo , NF-kappa B/metabolismoRESUMO
The effect of N2H4 (hydrazine) on AnAOB (anaerobic ammonia oxidizing bacteria) metabolic pattern is unknown. Therefore, the main purpose of this paper was to explore the effects of exogenous N2H4 on the SAA (specific anammox activity), characteristics and metabolic pathway of AnAOB. The results showed that low N2H4 concentration (1-5 mg/L) continuous dosing can promote SAA. The promoting effect was found to be more obvious within the dosage of 3-5 mg/L N2H4. It was also indicated that high N2H4 concentration dosing (5-10 mg/L) can trigger the self-protection mechanism of AnAOB granular sludge by secreting a large amount of B-PN (binding polymeric protein). Intermittent addition of N2H4 at low concentration is conducive to the long-term stable operation of anammox process. Exogenous N2H4 can be directly oxidized by AnAOB to promote the consumption of NO2--N and NH4+-N. In addition, excess electrons can also drive the process of NO3--N reduction and NO2--N disproportionation. Theoretically, these reaction processes need two and ten extra electrons respectively, which is not easy to occur compared with the anammox process.
Assuntos
Compostos de Amônio , Nitrogênio , Compostos de Amônio/metabolismo , Oxidação Anaeróbia da Amônia , Anaerobiose , Bactérias/metabolismo , Reatores Biológicos/microbiologia , Hidrazinas/metabolismo , Nitrogênio/metabolismo , Dióxido de Nitrogênio , Oxirredução , Esgotos/microbiologiaRESUMO
Ammonia-oxidizing archaea (AOA) and bacteria (AOB) perform key steps in the global nitrogen cycle, the oxidation of ammonia to nitrite. While the ammonia oxidation pathway is well characterized in AOB, many knowledge gaps remain about the metabolism of AOA. Hydroxylamine is an intermediate in both AOB and AOA, but homologues of hydroxylamine dehydrogenase (HAO), catalyzing bacterial hydroxylamine oxidation, are absent in AOA. Hydrazine is a substrate for bacterial HAO, while phenylhydrazine is a suicide inhibitor of HAO. Here, we examine the effect of hydrazines in AOA to gain insights into the archaeal ammonia oxidation pathway. We show that hydrazine is both a substrate and an inhibitor for AOA and that phenylhydrazine irreversibly inhibits archaeal hydroxylamine oxidation. Both hydrazine and phenylhydrazine interfered with ammonia and hydroxylamine oxidation in AOA. Furthermore, the AOA "Candidatus Nitrosocosmicus franklandus" C13 oxidized hydrazine into dinitrogen (N2), coupling this reaction to ATP production and O2 uptake. This study expands the known substrates of AOA and suggests that despite differences in enzymology, the ammonia oxidation pathways of AOB and AOA are functionally surprisingly similar. These results demonstrate that hydrazines are valuable tools for studying the archaeal ammonia oxidation pathway. IMPORTANCE Ammonia-oxidizing archaea (AOA) are among the most numerous living organisms on Earth, and they play a pivotal role in the global biogeochemical nitrogen cycle. Despite this, little is known about the physiology and metabolism of AOA. We demonstrate in this study that hydrazines are inhibitors of AOA. Furthermore, we demonstrate that the model soil AOA "Ca. Nitrosocosmicus franklandus" C13 oxidizes hydrazine to dinitrogen gas, and this reaction yields ATP. This provides an important advance in our understanding of the metabolism of AOA and expands the short list of energy-yielding compounds that AOA can use. This study also provides evidence that hydrazines can be useful tools for studying the metabolism of AOA, as they have been for the bacterial ammonia oxidizers.
Assuntos
Amônia , Archaea , Trifosfato de Adenosina/metabolismo , Amônia/metabolismo , Archaea/metabolismo , Bactérias/metabolismo , Humanos , Hidrazinas/metabolismo , Hidrazinas/farmacologia , Hidroxilaminas/metabolismo , Nitrificação , Fenil-Hidrazinas/metabolismo , Microbiologia do SoloRESUMO
The nitrogen-nitrogen bond is a core feature of diverse functional groups like hydrazines, nitrosamines, diazos, and pyrazoles. Such functional groups are found in >300 known natural products. Such N-N bond-containing functional groups are also found in significant percentage of clinical drugs. Therefore, there is wide interest in synthetic and enzymatic methods to form nitrogen-nitrogen bonds. In this review, we summarize synthetic and biosynthetic approaches to diverse nitrogen-nitrogen-bond-containing functional groups, with a focus on biosynthetic pathways and enzymes.
Assuntos
Produtos Biológicos , Nitrogênio , Produtos Biológicos/química , Vias Biossintéticas , Hidrazinas/química , Hidrazinas/metabolismo , Nitrogênio/químicaRESUMO
PURPOSE: Selinexor is an oral selective inhibitor of exportin-1 (XPO1) with efficacy in various solid and hematologic tumors. We assessed intratumoral penetration, safety, and efficacy of selinexor monotherapy for recurrent glioblastoma. PATIENTS AND METHODS: Seventy-six adults with Karnofsky Performance Status ≥ 60 were enrolled. Patients undergoing cytoreductive surgery received up to three selinexor doses (twice weekly) preoperatively (Arm A; n = 8 patients). Patients not undergoing surgery received 50 mg/m2 (Arm B, n = 24), or 60 mg (Arm C, n = 14) twice weekly, or 80 mg once weekly (Arm D; n = 30). Primary endpoint was 6-month progression-free survival rate (PFS6). RESULTS: Median selinexor concentrations in resected tumors from patients receiving presurgical selinexor was 105.4 nmol/L (range 39.7-291 nmol/L). In Arms B, C, and D, respectively, the PFS6 was 10% [95% confidence interval (CI), 2.79-35.9], 7.7% (95% CI, 1.17-50.6), and 17% (95% CI, 7.78-38.3). Measurable reduction in tumor size was observed in 19 (28%) and RANO-response rate overall was 8.8% [Arm B, 8.3% (95% CI, 1.0-27.0); C: 7.7% (95% CI, 0.2-36.0); D: 10% (95% CI, 2.1-26.5)], with one complete and two durable partial responses in Arm D. Serious adverse events (AEs) occurred in 26 (34%) patients; 1 (1.3%) was fatal. The most common treatment-related AEs were fatigue (61%), nausea (59%), decreased appetite (43%), and thrombocytopenia (43%), and were manageable by supportive care and dose modification. Molecular studies identified a signature predictive of response (AUC = 0.88). CONCLUSIONS: At 80 mg weekly, single-agent selinexor induced responses and clinically relevant PFS6 with manageable side effects requiring dose reductions. Ongoing trials are evaluating safety and efficacy of selinexor in combination with other therapies for newly diagnosed or recurrent glioblastoma.
Assuntos
Neoplasias Encefálicas/tratamento farmacológico , Glioblastoma/tratamento farmacológico , Hidrazinas/administração & dosagem , Recidiva Local de Neoplasia/tratamento farmacológico , Triazóis/administração & dosagem , Administração Oral , Adulto , Idoso , Encéfalo/metabolismo , Neoplasias Encefálicas/cirurgia , Procedimentos Cirúrgicos de Citorredução , Feminino , Glioblastoma/cirurgia , Humanos , Hidrazinas/efeitos adversos , Hidrazinas/metabolismo , Masculino , Pessoa de Meia-Idade , Resultado do Tratamento , Triazóis/efeitos adversos , Triazóis/metabolismo , Adulto JovemRESUMO
Laccase from pathogenic fungi participates in both the delignification and neutralization of phytoantibiotics. Furthermore, it interferes with the hormone signaling in plants and catalyzes melanization. Infections of these pathogens contribute to loss in forestry, agriculture, and horticulture. As there is still a need to expand knowledge on efficient defense strategies against phytopathogenic fungi, the present study aimed to reveal more information on the molecular mechanisms of laccase inhibition with natural and natural-like carboxylic acid semi-synthetic derivatives. A set of hydrazide-hydrazones derived from carboxylic acids, generally including electron-rich arene units that serve as a decoy substrate, was synthesized and tested with laccase from Trametes versicolor. The classic synthesis of the title inhibitors proceeded with good to almost quantitative yield. Ninety percent of the tested molecules were active in the range of KI = 8-233 µM and showed different types of action. Such magnitude of inhibition constants qualified the hydrazide-hydrazones as strong laccase inhibitors. Molecular docking studies supporting the experimental data explained the selected derivatives' interactions with the enzyme. The results are promising in developing new potential antifungal agents mitigating the damage scale in the plant cultivation, gardening, and horticulture sectors.
Assuntos
Inibidores Enzimáticos/farmacologia , Proteínas Fúngicas/antagonistas & inibidores , Hidrazinas/farmacologia , Lacase/antagonistas & inibidores , Fenóis/farmacologia , Polyporaceae/enzimologia , Biocatálise/efeitos dos fármacos , Domínio Catalítico , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Hidrazinas/química , Hidrazinas/metabolismo , Cinética , Lacase/química , Lacase/metabolismo , Modelos Químicos , Simulação de Acoplamento Molecular , Estrutura Molecular , Fenóis/química , Fenóis/metabolismo , Doenças das Plantas/microbiologia , Polyporaceae/patogenicidade , Relação Estrutura-AtividadeRESUMO
In mammalian testes, extensive remodeling of the microtubule (MT) and actin cytoskeletons takes place in Sertoli cells across the seminiferous epithelium to support spermatogenesis. However, the mechanism(s) involving regulatory and signaling proteins remains poorly understood. Herein, A-kinase anchoring protein 9 (AKAP9, a member of the AKAP multivalent scaffold protein family) was shown to be one of these crucial regulatory proteins in the rat testis. Earlier studies have shown that AKAP9 serves as a signaling platform by recruiting multiple signaling and regulatory proteins to create a large protein complex that binds to the Golgi and centrosome to facilitate the assembly of the MT-nucleating γ-tubulin ring complex to initiate MT polymerization. We further expanded our earlier studies based on a Sertoli cell-specific AKAP9 knockout mouse model to probe the function of AKAP9 by using the techniques of immunofluorescence analysis, RNA interference (RNAi), and biochemical assays on an in vitro primary Sertoli cell culture model, and an adjudin-based animal model. AKAP9 robustly expressed across the seminiferous epithelium in adult rat testes, colocalizing with MT-based tracks, and laid perpendicular across the seminiferous epithelium, and prominently expressed at the Sertoli-spermatid cell-cell anchoring junction (called apical ectoplasmic specialization [ES]) and at the Sertoli cell-cell interface (called basal ES, which together with tight junction [TJ] created the blood-testis barrier [BTB]) stage specifically. AKAP9 knockdown in Sertoli cells by RNAi was found to perturb the TJ-permeability barrier through disruptive changes in the distribution of BTB-associated proteins at the Sertoli cell cortical zone, mediated by a considerable loss of ability to induce both MT polymerization and actin filament bundling. A considerable decline in AKAP9 expression and a disruptive distribution of AKAP9 across the seminiferous tubules was also noted during adjudin-induced germ cell (GC) exfoliation in this animal model, illustrating AKAP9 is essential to maintain the homeostasis of cytoskeletons to maintain Sertoli and GC adhesion in the testis.
Assuntos
Proteínas de Ancoragem à Quinase A/metabolismo , Citoesqueleto de Actina/metabolismo , Proteínas do Citoesqueleto/metabolismo , Microtúbulos/metabolismo , Espermatogênese , Testículo/citologia , Testículo/metabolismo , Animais , Núcleo Celular/metabolismo , Hidrazinas/metabolismo , Indazóis/metabolismo , Masculino , Modelos Animais , Ratos , Células de Sertoli/citologia , Células de Sertoli/metabolismo , Testículo/químicaRESUMO
N-hydroxylating monooxygenases (NMOs) are a subclass of flavin-dependent enzymes that hydroxylate nitrogen atoms. Recently, unique NMOs that perform multiple reactions on one substrate molecule have been identified. Fosfazinomycin M (FzmM) is one such NMO, forming nitrosuccinate from aspartate (Asp) in the fosfazinomycin biosynthetic pathway in some Streptomyces sp. This work details the biochemical and kinetic analysis of FzmM. Steady-state kinetic investigation shows that FzmM performs a coupled reaction with Asp (kcat, 3.0 ± 0.01 s-1) forming nitrosuccinate, which can be converted to fumarate and nitrite by the action of FzmL. FzmM displays a 70-fold higher kcat/KM value for NADPH compared to NADH and has a narrow optimal pH range (7.5-8.0). Contrary to other NMOs where the kred is rate-limiting, FzmM exhibits a very fast kred (50 ± 0.01 s-1 at 4 °C) with NADPH. NADPH binds at a KD value of â¼400 µM, and hydride transfer occurs with pro-R stereochemistry. Oxidation of FzmM in the absence of Asp exhibits a spectrum with a shoulder at â¼370 nm, consistent with the formation of a C(4a)-hydroperoxyflavin intermediate, which decays into oxidized flavin and hydrogen peroxide at a rate 100-fold slower than the kcat. This reaction is enhanced in the presence of Asp with a slightly faster kox than the kcat, suggesting that flavin dehydration or Asp oxidation is partially rate limiting. Multiple sequence analyses of FzmM to NMOs identified conserved residues involved in flavin binding but not for NADPH. Additional sequence analysis to related monooxygenases suggests that FzmM shares sequence motifs absent in other NMOs.
Assuntos
Hidrazinas/metabolismo , Compostos Organofosforados/metabolismo , Dinitrocresóis , Flavina-Adenina Dinucleotídeo/metabolismo , Flavinas/metabolismo , Hidroxilação/fisiologia , Cinética , Oxigenases de Função Mista/metabolismo , NADP/metabolismo , Oxirredução , Ácido Succínico/metabolismoRESUMO
Four new transition metal complexes, [M(PPh3)(L)].CH3OH (M = Ni(II) (1), Pd(II) (2)) [Pt (PPh3)2(HL)]Cl (3) and [Ru(CO)(PPh3)2(L)] (4) (H2L = 2,4-dihydroxybenzaldehyde-S-methyldithiocarbazate, PPh3 = triphenylphosphine) have been synthesized and characterized by elemental analyses (C, H, N), FTIR, NMR (1H, 31P), ESI-MS and UV-visible spectroscopy. The molecular structure of (1) and (2) complexes was confirmed by single-crystal X-ray crystallography. It showed a distorted square planar geometry for both complexes around the metal center, and the H2L adopt a bi-negative tridentate chelating mode. The interaction with biomolecules viz., calf thymus DNA (ct DNA), yeast RNA (tRNA), and BSA (bovine serum albumin) was examined by both UV-visible and fluorescence spectroscopies. The antioxidant activity of all compounds is discussed on basis of DPPH⢠(2,2-diphenyl-1-picrylhydrazyl) scavenging activity and showed better antioxidant activity for complexes compared to the ligand. The in vitro cytotoxicity of the compounds was tested on human (breast cancer (MCF7), colon cancer (HCT116), liver cancer (HepG2), and normal lung fibroblast (WI38)) cell lines, showing that complex (1) the most potent against MCF7 and complex (4) against HCT116 cell lines based on IC50 and selective indices (SI) values. So, both complexes were chosen for further studies such as DNA fragmentation, cell apoptosis, and cell cycle analyses. Complex (1) induced MCF7 cell death by cellular apoptosis and arrest cells at S phase. Complex (4) induced HCT116 cell death predominantly by cellular necrosis and arrested cell division at G2/M phase due to DNA damage.
Assuntos
Antineoplásicos/farmacologia , Complexos de Coordenação/farmacologia , Sequestradores de Radicais Livres/farmacologia , Hidrazinas/farmacologia , Fosfinas/farmacologia , Tiocarbamatos/farmacologia , Animais , Antineoplásicos/síntese química , Antineoplásicos/metabolismo , Apoptose/efeitos dos fármacos , Bovinos , Linhagem Celular Tumoral , Complexos de Coordenação/síntese química , Complexos de Coordenação/metabolismo , DNA/metabolismo , Fragmentação do DNA/efeitos dos fármacos , Sequestradores de Radicais Livres/síntese química , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Humanos , Hidrazinas/síntese química , Hidrazinas/metabolismo , Metais Pesados/química , Fosfinas/síntese química , Fosfinas/metabolismo , Ligação Proteica , RNA de Transferência/metabolismo , Pontos de Checagem da Fase S do Ciclo Celular/efeitos dos fármacos , Soroalbumina Bovina/metabolismo , Tiocarbamatos/síntese química , Tiocarbamatos/metabolismo , Leveduras/químicaRESUMO
The novel coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) still has serious negative effects on health, social life, and economics. Recently, vaccines from various companies have been urgently approved to control SARS-CoV-2 infections. However, any specific antiviral drug has not been confirmed so far for regular treatment. An important target is the main protease (Mpro ), which plays a major role in replication of the virus. In this study, Gaussian and residue network models are employed to reveal two distinct potential allosteric sites on Mpro that can be evaluated as drug targets besides the active site. Then, Food and Drug Administration (FDA)-approved drugs are docked to three distinct sites with flexible docking using AutoDock Vina to identify potential drug candidates. Fourteen best molecule hits for the active site of Mpro are determined. Six of these also exhibit high docking scores for the potential allosteric regions. Full-atom molecular dynamics simulations with MM-GBSA method indicate that compounds docked to active and potential allosteric sites form stable interactions with high binding free energy (∆Gbind ) values. ∆Gbind values reach -52.06 kcal/mol for the active site, -51.08 kcal/mol for the potential allosteric site 1, and - 42.93 kcal/mol for the potential allosteric site 2. Energy decomposition calculations per residue elucidate key binding residues stabilizing the ligands that can further serve to design pharmacophores. This systematic and efficient computational analysis successfully determines ivermectine, diosmin, and selinexor currently subjected to clinical trials, and further proposes bromocriptine, elbasvir as Mpro inhibitor candidates to be evaluated against SARS-CoV-2 infections.
Assuntos
Antivirais/metabolismo , Benzofuranos/química , Proteases 3C de Coronavírus/metabolismo , Reposicionamento de Medicamentos/métodos , Imidazóis/química , Sítio Alostérico , Antivirais/química , Antivirais/farmacologia , Benzofuranos/metabolismo , Benzofuranos/farmacologia , Sítios de Ligação , Bromocriptina/química , Bromocriptina/metabolismo , Bromocriptina/farmacologia , Proteases 3C de Coronavírus/antagonistas & inibidores , Proteases 3C de Coronavírus/química , Diosmina/química , Diosmina/metabolismo , Hidrazinas/química , Hidrazinas/metabolismo , Hidrazinas/farmacologia , Imidazóis/metabolismo , Imidazóis/farmacologia , Ivermectina/química , Ivermectina/metabolismo , Ivermectina/farmacologia , Ligantes , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Triazóis/química , Triazóis/metabolismo , Triazóis/farmacologia , Estados Unidos , United States Food and Drug AdministrationRESUMO
Triple negative breast cancer (TNBC) has a worse prognosis than other types of breast cancer due to its special biological behavior and clinicopathological characteristics. TNBC cell proliferation and progression to metastasis can be suppressed by inducing cytostatic autophagy. mTOR is closely related to autophagy and is involved in protein synthesis, nutrient metabolism and activating mTOR promotes tumor growth and metastasis. In this paper, we adopted the strategy of structure simplification, aimed to look for novel small-molecule inhibitors of mTOR by pharmacophore-based virtual screening and biological activity determination. We found a lead compound with 3-bromo-N'-(4-hydroxybenzylidene)-4-methylbenzohydrazide for rational drug design and structural modification, then studied its structure-activity relationship. After that, compound 7c with the best TNBC cells inhibitory activities and superior mTOR enzyme inhibitory activity was obtained. In addition, we found that compound 7c could induce autophagic cell death and apoptosis in MDA-MB-231 and MDA-MB-468 cell lines. In conclusion, these findings provide new clues for our 3-bromo-N'-(4-hydroxybenzylidene)-4-methylbenzohydrazide derivatives, which are expected to become drug candidates for the treatment of TNBC in the future.
Assuntos
Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Hidrazinas/química , Inibidores de Proteínas Quinases/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Sítios de Ligação , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Hidrazinas/metabolismo , Hidrazinas/farmacologia , Simulação de Acoplamento Molecular , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/metabolismo , Relação Estrutura-Atividade , Serina-Treonina Quinases TOR/metabolismo , Neoplasias de Mama Triplo Negativas/patologiaRESUMO
Anaerobic Ammonium Oxidation ("anammox") is a bacterial process in which nitrite and ammonium are converted into nitrogen gas and water, yielding energy for the cell. Anammox is an important branch of the global biological nitrogen cycle, being responsible for up to 50% of the yearly nitrogen removal from the oceans. Strikingly, the anammox process uniquely relies on the extremely reactive and toxic compound hydrazine as a free intermediate. Given its global importance and biochemical novelty, there is considerable interest in the enzymes at the heart of the anammox pathway. Unfortunately, obtaining these enzymes in sufficiently large amounts for biochemical and structural studies is problematic, given the slow growth of pure cultures of anammox bacteria when high cell densities are required. However, the anammox process is being applied in wastewater treatment to remove nitrogenous waste in processes like DEamMONification (DEMON). In plants using such processes, which rely on a combination of aerobic ammonia-oxidizers and anammox organisms, kilogram amounts of anammox bacteria-containing sludge are readily available. Here, we report a protein isolation protocol starting from anammox cells present in DEMON sludge from a wastewater treatment plan that readily yields pure preparations of key anammox proteins in the tens of milligrams, including hydrazine synthase HZS and hydrazine dehydrogenase (HDH), as well as hydroxylamine oxidoreductase (HAO). HDH and HAO were active and of sufficient quality for biochemical studies and for HAO, the crystal structure could be determined. The method presented here provides a viable way to obtain materials for the study of proteins not only from the central anammox metabolism but also for the study of other exciting aspects of anammox bacteria, such as for example, their unusual ladderane lipids.