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1.
Pharmaceuticals (Basel) ; 16(12)2023 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-38139821

RESUMO

Candida albicans and non-albicans Candida species are a common cause of human mucosal infections, as well as bloodstream infections and deep mycoses. The emergence of resistance of Candida spp. to antifungal drugs used in practice requires the search for new antimycotics. The present study unravels the antifungal potential of the synthetic dialk(en)ylthiosulfinates in comparison with an enzymatic in situ methionine γ-lyase-based thiosulfinate generation system (TGS). The kinetics of the TGS reaction, namely, the methionine γ-lyase-catalyzed ß-elimination of S-alk(en)yl-L-cysteine sulfoxides, was investigated via 1H NMR spectroscopy for the first time, revealing fast conversion rates and the efficient production of anticandidal dialk(en)ylthiosulfinates. The anticandidal potential of this system vs. synthetic thiosulfinates was investigated through an in vitro assay. TGS proved to be more effective (MIC range 0.36-1.1 µg/mL) than individual substances (MIC range 0.69-3.31 µg/mL). The tested preparations had an additive effect with the commercial antimycotics fluconazole, amphotericin B and 5-flucytosine demonstrating a fractional inhibitory coefficient index in the range of 0.5-2 µg/mL. TGS can be regarded as an attractive candidate for the targeted delivery of antimycotic thiosulfinates and for further implementation onto medically implanted devices.

2.
Biochemistry (Mosc) ; 88(7): 912-923, 2023 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-37751863

RESUMO

Pharmacological value of some natural compounds makes them attractive for use in oncology. The sulfur-containing thiosulfinates found in plants of the genus Allium have long been known as compounds with various therapeutic properties, including antitumor. Over the last few years, the effect of thiosulfinates on various stages of carcinogenesis has been actively investigated. In vitro and in vivo studies have shown that thiosulfinates inhibit proliferation of cancer cells, as well as they induce apoptosis. The purpose of this review is to summarize current data on the use of natural and synthetic thiosulfinates in cancer therapy. Antitumor mechanisms and molecular targets of these promising compounds are discussed. A significant part of the review is devoted to consideration of a new strategy for treatment of oncological diseases - use of the directed enzyme prodrug therapy approach aiming to obtain antitumor thiosulfinates in situ.

3.
Biochemistry (Mosc) ; 88(5): 600-609, 2023 May.
Artigo em Inglês | MEDLINE | ID: mdl-37331706

RESUMO

O-acetylhomoserine sulfhydrylase is one of the key enzymes in biosynthesis of methionine in Clostridioides difficile. The mechanism of γ-substitution reaction of O-acetyl-L-homoserine catalyzed by this enzyme is the least studied among the pyridoxal-5'-phosphate-dependent enzymes involved in metabolism of cysteine and methionine. To clarify the role of active site residues Tyr52 and Tyr107, four mutant forms of the enzyme with replacements of these residues with phenylalanine and alanine were generated. Catalytic and spectral properties of the mutant forms were investigated. The rate of γ-substitution reaction catalyzed by the mutant forms with replaced Tyr52 residue decreased by more than three orders of magnitude compared to the wild-type enzyme. The Tyr107Phe and Tyr107Ala mutant forms practically did not catalyze this reaction. Replacements of the Tyr52 and Tyr107 residues led to the decrease in affinity of apoenzyme to coenzyme by three orders of magnitude and changes in the ionic state of the internal aldimine of the enzyme. The obtained results allowed us to assume that Tyr52 is involved in ensuring optimal position of the catalytic coenzyme-binding lysine residue at the stages of C-α-proton elimination and elimination of the side group of the substrate. Tyr107 could act as a general acid catalyst at the stage of acetate elimination.


Assuntos
Clostridioides difficile , Clostridioides difficile/metabolismo , Cisteína Sintase/química , Cisteína Sintase/metabolismo , Domínio Catalítico , Clostridioides/metabolismo , Tirosina , Fosfato de Piridoxal/química , Fosfato de Piridoxal/metabolismo , Metionina , Cinética
4.
Front Plant Sci ; 14: 1336192, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-38283969

RESUMO

Introduction: Pubescence is an important phenotypic trait observed in both vegetative and generative plant organs. Pubescent plants demonstrate increased resistance to various environmental stresses such as drought, low temperatures, and pests. It serves as a significant morphological marker and aids in selecting stress-resistant cultivars, particularly in wheat. In wheat, pubescence is visible on leaves, leaf sheath, glumes and nodes. Regarding glumes, the presence of pubescence plays a pivotal role in its classification. It supplements other spike characteristics, aiding in distinguishing between different varieties within the wheat species. The determination of pubescence typically involves visual analysis by an expert. However, methods without the use of binocular loupe tend to be subjective, while employing additional equipment is labor-intensive. This paper proposes an integrated approach to determine glume pubescence presence in spike images captured under laboratory conditions using a digital camera and convolutional neural networks. Methods: Initially, image segmentation is conducted to extract the contour of the spike body, followed by cropping of the spike images to an equal size. These images are then classified based on glume pubescence (pubescent/glabrous) using various convolutional neural network architectures (Resnet-18, EfficientNet-B0, and EfficientNet-B1). The networks were trained and tested on a dataset comprising 9,719 spike images. Results: For segmentation, the U-Net model with EfficientNet-B1 encoder was chosen, achieving the segmentation accuracy IoU = 0.947 for the spike body and 0.777 for awns. The classification model for glume pubescence with the highest performance utilized the EfficientNet-B1 architecture. On the test sample, the model exhibited prediction accuracy parameters of F1 = 0.85 and AUC = 0.96, while on the holdout sample it showed F1 = 0.84 and AUC = 0.89. Additionally, the study investigated the relationship between image scale, artificial distortions, and model prediction performance, revealing that higher magnification and smaller distortions yielded a more accurate prediction of glume pubescence.

5.
Int J Mol Sci ; 23(19)2022 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-36233347

RESUMO

The purpose of this study was to determine the anticancer effect of dipropyl thiosulfinate produced in situ by the pharmacological pair: (1) conjugated with daidzein C115H methionine γ-lyase (EC 4.4.1.11, C115H MGL-Dz) and (2) the substrate, S-propyl-L-cysteine sulfoxide (propiin) against various solid tumor types in vitro and in vivo. The MTT test was used to calculate IC50 values for HT29, COLO205 and HCT116 (colon cancer); Panc1 and MIA-PaCa2 (pancreatic cancer); and 22Rv1, DU-145 and PC3 (prostate cancer). The most promising effect for colon cancer cells in vitro was observed in HT29 (IC50 = 6.9 µM). The IC50 values for MIA-PaCa2 and Panc1 were 3.4 and 3.8 µM, respectively. Among prostate cancer cells, 22Rv1 was the most sensitive (IC50 = 5.4 µM). In vivo antitumor activity of the pharmacological pair was studied in HT29, SW620, Panc1, MIA-PaCa2 and 22Rv1 subcutaneous xenografts in BALB/c nude mice. The application of C115H MGL-Dz /propiin demonstrated a significant reduction in the tumor volume of Panc1 (TGI 67%; p = 0.004), MIA-PaCa2 (TGI 50%; p = 0.011), HT29 (TGI 51%; p = 0.04) and 22Rv1 (TGI 70%; p = 0.043) xenografts. The results suggest that the combination of C115H MGL-Dz/propiin is able to suppress tumor growth in vitro and in vivo and the use of this pharmacological pair can be considered as a new strategy for the treatment of solid tumors.


Assuntos
Neoplasias do Colo , Neoplasias Pancreáticas , Pró-Fármacos , Neoplasias da Próstata , Animais , Liases de Carbono-Enxofre , Linhagem Celular Tumoral , Cisteína/análogos & derivados , Xenoenxertos , Humanos , Isoflavonas , Masculino , Metionina , Camundongos , Camundongos Nus , Neoplasias da Próstata/tratamento farmacológico , Sulfóxidos
6.
Biochimie ; 194: 13-18, 2022 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-34923045

RESUMO

Therapeutic enzymes used for the treatment of a wide range of human disorders often suffer from suboptimal pharmacokinetics and stability. Engineering approaches such as encapsulation in micro- and nanocarriers, and replacements of amino acid residues of the native enzyme provide significant potential for improving the performance of enzyme therapy. Here, we develop a nanodelivery system on the base of polyion complex vesicles (PICsomes) that includes methionine γ-lyase (MGL) as a therapeutic enzyme. We have two strategies for using the enzyme: first, methionine γ-lyase is an anticancer agent removing l-methionine from plasma, second, the binary system methionine γ-lyase/S-alk(en)yl-l-cysteine sulfoxides is effective in enzyme prodrug therapy (EPT). Various lengths polymers were synthesized, and two mutant forms of the enzyme were used. The catalytic and pharmacokinetic parameters of the nanoformulations were investigated. The catalytic efficiencies of encapsulated enzymes were comparable to that of native enzymes. Pharmacokinetic analysis has shown that inclusion into PICsomes increases half-life of the enzymes, and they can be safely administered in vivo. The results suggest the further use of encapsulated MGLs for EPT and anticancer therapy, and this strategy could be leveraged to improve the efficiency of enzyme-based therapies for managing serious human diseases.


Assuntos
Liases , Liases de Carbono-Enxofre/metabolismo , Cisteína/química , Humanos , Cinética , Liases/metabolismo , Metionina/metabolismo , Sulfóxidos/metabolismo
7.
Protein Expr Purif ; 180: 105810, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33338587

RESUMO

The gene NT01CX_1210 of pathogenic bacterium Clostridium novyi annotated as encoding O-acetylhomoserine sulfhydrylase was cloned and expressed in Escherichia coli. The gene product having O-acetylhomoserine sulfhydrylase activity was purified to homogeneity. The protein showed molecular mass of approximately 184 kDa for the native form and 46 kDa for the subunit. The enzyme catalyzes the γ-substitution reaction of O-acetylhomoserine with maximum activity at pH 7.5. Analysis of C. novyi genome allowed us to suggest that there is only one way for the synthesis of l-methionine in the bacterium. The data obtained may provide the basis for further study of the role of OAHS in Clostridium bacteria and an ascertainment of its mechanism.


Assuntos
Proteínas de Bactérias , Carbono-Oxigênio Liases , Clonagem Molecular , Clostridium/genética , Expressão Gênica , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Carbono-Oxigênio Liases/biossíntese , Carbono-Oxigênio Liases/química , Carbono-Oxigênio Liases/genética , Carbono-Oxigênio Liases/isolamento & purificação , Clostridium/enzimologia , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação
8.
Bioorg Med Chem ; 28(7): 115378, 2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-32089391

RESUMO

A set of AT-specific fluorescent dimeric bisbenzimidazoles DBPA(n) with linkers of different lengths bound to DNA in the minor groove were synthesized and their genetic, virological, and biochemical studies were performed. The DBPA(n) were shown to be effective inhibitors of the histon-like protein H-NS, a regulator of the DNA transcription factor, as well as of the Aliivibrio logei Quorum Sensing regulatory system in E. coli cells. Their antiviral activity was tested in model cell lines infected with herpes simplex virus type I. Also, it was found that DBPA(n) could inhibit catalytic activities of HIV-1 integrase at low micromolar concentrations. All of the dimeric bisbenzimidazoles DBPA(n) manifested fluorescent properties, were well soluble in water, nontoxic up to concentrations of 200 µM, and could penetrate into nuclei followed by binding to DNA.


Assuntos
Bisbenzimidazol/química , Bisbenzimidazol/farmacologia , DNA/química , Aliivibrio/metabolismo , Antibacterianos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Sequência de Bases , DNA/genética , Desenho de Fármacos , Escherichia coli/metabolismo , Corantes Fluorescentes , Integrase de HIV , Inibidores de Integrase de HIV/farmacologia , Ligantes , Estrutura Molecular , Pirróis , Percepção de Quorum/fisiologia , Relação Estrutura-Atividade
9.
Biochimie ; 168: 190-197, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31711941

RESUMO

Interactions of Citrobacter freundii methionine γ-lyase (MGL) with sulfoxides of typical substrates were investigated. It was found that sulfoxides are suicide substrates of the enzyme. The products of the ß- and γ-elimination reactions of sulfoxides, thiosulfinates, oxidize three cysteine residues of the enzyme. Three-dimensional structures of MGL inactivated by dimethyl thiosulfinate and diethyl thiosulfinate were determined at 1.46 Šand 1.59 Šresolution. Analysis of the structures identified SH groups oxidized by thiosulfinates and revealed the structural bases of MGL inactivation. The extent of inactivation of MGL in the catalysis of the ß-elimination reaction depends on the length of the «tail¼ at oxidized Cys115. Oxidation of Cys115 results in MGL incapable to catalyze the stage of methyl mercaptan elimination of the physiological reaction.


Assuntos
Aminoácidos/química , Liases de Carbono-Enxofre/química , Citrobacter freundii/enzimologia , Cisteína/química , Sulfóxidos/química , Proteínas de Bactérias/química , Cinética , Ligantes , Modelos Moleculares
10.
IUBMB Life ; 71(11): 1815-1823, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31359602

RESUMO

O-acetylhomoserine sulfhydrylase (OAHS) is a pyridoxal 5'-phosphate-dependent enzyme involved in microbial methionine biosynthesis. In this study, we report gene cloning, protein purification, and some biochemical characteristics of OAHS from Clostridioides difficile. The enzyme is a tetramer with molecular weight of 185 kDa. It possesses a high activity in the reaction of L-homocysteine synthesis, comparable to reported activities of OAHSes from other sources. OAHS activity is inhibited by metabolic end product L-methionine. L-Propargylglycine was found to be a suicide inhibitor of the enzyme. Substrate analogue Nγ -acetyl-L-2,4-diaminobutyric acid is a competitive inhibitor of OAHS with Ki = 0.04 mM. Analysis of C. difficile genome allows to suggest that the bacterium uses the way of direct sulfhydrylation for the synthesis of L-methionine. The data obtained may provide the basis for further study of the role of OAHS in the pathogenic bacterium and the development of potential inhibitors.


Assuntos
Alcinos/metabolismo , Carbono-Oxigênio Liases/metabolismo , Clonagem Molecular/métodos , Clostridioides difficile/enzimologia , Glicina/análogos & derivados , Metionina/biossíntese , Fosfato de Piridoxal/metabolismo , Compostos de Sulfidrila/metabolismo , Sequência de Aminoácidos , Carbono-Oxigênio Liases/genética , Clostridioides difficile/genética , Genoma Bacteriano , Glicina/metabolismo , Homologia de Sequência , Especificidade por Substrato
11.
Bioorg Med Chem ; 26(9): 2302-2309, 2018 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-29602675

RESUMO

A series of DNA minor groove binding fluorescent dimeric bisbenzimidazoles DBA(n) bearing linkers of various length were synthesized and their biochemical and antiviral activities were evaluated. Their antiviral activity was assessed in model cell systems infected with human herpes simplex virus (HSV-1) and cytomegalovirus (CMV). Compounds DBA(1) and DBA(7) demonstrated in vitro inhibitory properties towards HSV-1, and DBA(7) completely blocked the viral infection. Compound DBA(11) displayed the in vitro therapeutic activity towards both HSV-1 and CMV. All of the DBA(n) could fluoresce, were well soluble in water, not cytotoxic to a concentration of 240 µM, penetrated well into cell nuclei by binding to DNA and could inhibit topo-I at low micromolecular concentrations.


Assuntos
Antivirais/química , Benzimidazóis/química , DNA/química , Animais , Antivirais/síntese química , Antivirais/toxicidade , Benzimidazóis/síntese química , Benzimidazóis/toxicidade , Bovinos , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Chlorocebus aethiops , Citomegalovirus/efeitos dos fármacos , Fluorescência , Herpesvirus Humano 1/efeitos dos fármacos , Humanos , Ligantes , Solubilidade , Inibidores da Topoisomerase I/síntese química , Inibidores da Topoisomerase I/química , Inibidores da Topoisomerase I/toxicidade , Células Vero
12.
PLoS One ; 13(1): e0189826, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29329300

RESUMO

BACKGROUND: Hypermethylation is observed in the promoter regions of suppressor genes in the tumor cancer cells. Reactivation of these genes by demethylation of their promoters is a prospective strategy of the anticancer therapy. Previous experiments have shown that symmetric dimeric bisbenzimidazoles DBP(n) are able to block DNA methyltransferase activities. It was also found that DBP(n) produces a moderate effect on the activation of total gene expression in HeLa-TI population containing epigenetically repressed avian sarcoma genome. PRINCIPAL FINDINGS: It is shown that DBP(n) are able to penetrate the cellular membranes and accumulate in breast carcinoma cell MCF-7, mainly in the mitochondria and in the nucleus, excluding the nucleolus. The DBP(n) are non-toxic to the cells and have a weak overall demethylation effect on genomic DNA. DBP(n) demethylate the promoter regions of the tumor suppressor genes PTEN and RARB. DBP(n) promotes expression of the genes RARB, PTEN, CDKN2A, RUNX3, Apaf-1 and APC "silent" in the MCF-7 because of the hypermethylation of their promoter regions. Simultaneously with the demethylation of the DNA in the nucleus a significant increase in the methylation level of rRNA genes in the nucleolus was detected. Increased rDNA methylation correlated with a reduction of the rRNA amount in the cells by 20-30%. It is assumed that during DNA methyltransferase activity inhibition by the DBP(n) in the nucleus, the enzyme is sequestered in the nucleolus and provides additional methylation of the rDNA that are not shielded by DBP(n). CONCLUSIONS/SIGNIFICANCE: It is concluded that DBP (n) are able to accumulate in the nucleus (excluding the nucleolus area) and in the mitochondria of cancer cells, reducing mitochondrial potential. The DBP (n) induce the demethylation of a cancer cell's genome, including the demethylation of the promoters of tumor suppressor genes. DBP (n) significantly increase the methylation of ribosomal RNA genes in the nucleoli. Therefore the further study of these compounds is needed; it could lead to the creation of new anticancer agents.


Assuntos
Benzimidazóis/farmacologia , Metilação de DNA/efeitos dos fármacos , RNA Ribossômico/genética , Receptores do Ácido Retinoico/genética , Benzimidazóis/química , Dimerização , Células HeLa , Humanos , Células MCF-7 , PTEN Fosfo-Hidrolase , Espécies Reativas de Oxigênio/metabolismo
13.
Biochim Biophys Acta Proteins Proteom ; 1865(9): 1123-1128, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28602917

RESUMO

The mutant form of Citrobacter freundii methionine γ-lyase with the replacement of active site Cys115 for His has been found to be inactive in the γ-elimination reaction of methionine while fully active in the γ-elimination reaction of O-acetyl-l-homoserine and in the ß-elimination reaction of S-alk(en)yl-substituted cysteines. In this work, the crystal structure of the mutant enzyme complexed with competitive inhibitor, l-norleucine was determined at 1.45Å resolution. At the enzyme active site the inhibitor proved to be bound both noncovalently and covalently, which corresponds to the two intermediates of the γ- and ß-elimination reactions, Michaelis complex and the external aldimine. Analysis of the structure allowed us to suggest the possible reason for the inability of the mutant enzyme to catalyze the physiological reaction.


Assuntos
Proteínas de Bactérias/química , Liases de Carbono-Enxofre/química , Citrobacter freundii/enzimologia , Mutação de Sentido Incorreto , Norleucina/metabolismo , Mutação Puntual , Substituição de Aminoácidos , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/metabolismo , Liases de Carbono-Enxofre/antagonistas & inibidores , Liases de Carbono-Enxofre/metabolismo , Domínio Catalítico , Citrobacter freundii/genética , Cristalografia por Raios X , Modelos Moleculares , Ligação Proteica , Conformação Proteica
14.
Bioorg Med Chem Lett ; 25(13): 2634-8, 2015 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-25987376

RESUMO

A series of new fluorescent symmetric dimeric bisbenzimidazoles DBP(n) bearing bisbenzimidazole fragments joined by oligomethylene linkers with a central 1,4-piperazine residue were synthesized. The complex formation of DBP(n) in the DNA minor groove was demonstrated. The DBP(n) at micromolar concentrations inhibit in vitro eukaryotic DNA topoisomerase I and prokaryotic DNA methyltransferase (MTase) M.SssI. The DBP(n) were soluble well in aqueous solutions and could penetrate cell and nuclear membranes and stain DNA in live cells. The DBP(n) displayed a moderate effect on the reactivation of gene expression.


Assuntos
Bisbenzimidazol/análogos & derivados , DNA/química , DNA/efeitos dos fármacos , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/farmacologia , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Antineoplásicos/farmacologia , Bisbenzimidazol/síntese química , Bisbenzimidazol/farmacologia , Linhagem Celular , DNA/genética , DNA-Citosina Metilases/antagonistas & inibidores , Dimerização , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Corantes Fluorescentes/química , Expressão Gênica/efeitos dos fármacos , Humanos , Células MCF-7 , Camundongos , Microscopia de Fluorescência , Relação Estrutura-Atividade , Inibidores da Topoisomerase I/síntese química , Inibidores da Topoisomerase I/química , Inibidores da Topoisomerase I/farmacologia
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