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1.
Chem Biol Interact ; 345: 109536, 2021 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-34058176

RESUMO

In this study, seven new 4-oxothiazolidine derivatives were synthesized and assayed, along 7 known derivatives, for inhibitory properties against deoxyribonuclease I (DNase I) and xanthine oxidase (XO) in vitro. Among tested compounds, (5Z)-Ethyl-2-(2-(cyanomethylene)-4-oxothiazolidin-5-yliden)acetate (6) exhibited inhibitory activity against both enzymes (DNase I IC50 = 67.94 ± 5.99 µM; XO IC50 = 98.98 ± 13.47 µM), therefore being the first reported dual inhibitor of DNase I and XO. Observed DNase I inhibition qualifies compound 6 as the most potent small organic DNase I inhibitor reported so far. Derivatives of 2-alkyliden-4-oxothiazolidinone (1) inhibited DNase I below 200 µM, while the other tested 4-oxothiazolidine derivatives remained inactive against both enzymes. The molecular docking and molecular dynamics simulations into the binding sites of DNase I and XO enzyme allowed us to clarify the binding modes of this 4-oxothiazolidine derivative, which might aid future development of dual DNase I and XO.


Assuntos
Desoxirribonuclease I/antagonistas & inibidores , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Tiazolidinas/síntese química , Tiazolidinas/farmacologia , Xantina Oxidase/antagonistas & inibidores , Técnicas de Química Sintética , Desoxirribonuclease I/química , Desoxirribonuclease I/metabolismo , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Conformação Proteica , Tiazolidinas/química , Tiazolidinas/metabolismo , Xantina Oxidase/química , Xantina Oxidase/metabolismo
2.
Bioorg Med Chem Lett ; 32: 127718, 2021 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-33253880

RESUMO

The search for new antimicrobial agents is greater than ever due to the perpetual threat of multidrug resistance in known pathogens and the relentless emergence of new infections. In this manuscript, ten thiazole-based thiazolidinone hybrids bearing a 6-trifluoromethoxy substituent on the benzothiazole core were synthesized and evaluated against a panel of four bacterial strains Salmonella typhimurium, Staphylococcus aureus, Escherichia coli and Listeria monocytogenes and three resistant strains Pseudomonas aeruginosa, E. coli and MRSA. The evaluation of minimum bactericidal and minimum inhibitory concentrations was accomplished by microdilution assay. As reference compounds ampicillin and streptomycin were employed. All compounds displayed antibacterial efficiencies with MBCs/MICs at 0.25-1 mg/mL and 0.12-1 mg/mL respectively while ampicillin displayed MBCs/MICs at 0.15-0.3 mg/mL and at 0.1-0.2 mg/mL respectively. MICs/MBC of streptomycin varied from 0.05 to 0.15 mg/mL and from 0.1 to 0.3 mg/mL respectively. The best overall effect was observed for compound h4, while compound h1 exhibited the highest effective action against E. coli (MIC/MBC 0.12/0.25 mg/ml) among all tested compounds.


Assuntos
Anti-Infecciosos/síntese química , Tiazóis/química , Tiazolidinas/química , Anti-Infecciosos/metabolismo , Anti-Infecciosos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Desidrogenases de Carboidrato/antagonistas & inibidores , Desidrogenases de Carboidrato/metabolismo , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Isomerismo , Testes de Sensibilidade Microbiana , Simulação de Acoplamento Molecular , Tiazolidinas/metabolismo , Tiazolidinas/farmacologia
3.
Bioorg Med Chem Lett ; 30(23): 127549, 2020 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-32927029

RESUMO

Metronidazole and its derivatives are widely used for the treatment of amoebiasis. However, metronidazole is considered as the standard drug but it has many side effects. The present study describes the synthesis of a series of metronidazole based thiazolidinone analogs via Knoevenagel condensation of 4-[2-(2-methyl-5-nitro-1H-imidazole-1-yl)ethoxy]benzaldehyde 1 with various thiazolidinone derivatives 2-14 to get the new scaffold (15-27) having better activity and lesser toxicity. Six compounds have shown better efficacy and lesser cytotoxicity than the standard drug metronidazole towards HM1: IMSS strain of Entamoeba histolytica. These compounds may combat the problem of drug resistance and might be effective in identifying potential alternatives for future drug discovery against EhOASS.


Assuntos
Amebicidas/farmacologia , Metronidazol/farmacologia , Tiazolidinas/farmacologia , Amebicidas/síntese química , Amebicidas/metabolismo , Amebicidas/toxicidade , Domínio Catalítico , Entamoeba histolytica/efeitos dos fármacos , Células HEK293 , Humanos , Metronidazol/síntese química , Metronidazol/metabolismo , Metronidazol/toxicidade , Simulação de Acoplamento Molecular , Estrutura Molecular , Testes de Sensibilidade Parasitária , Ligação Proteica , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Relação Quantitativa Estrutura-Atividade , Sulfatases/química , Sulfatases/metabolismo , Tiazolidinas/síntese química , Tiazolidinas/metabolismo , Tiazolidinas/toxicidade
4.
Anal Bioanal Chem ; 412(27): 7535-7546, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32840653

RESUMO

A novel method for the quantification of the sulfur-containing metabolites of formaldehyde (thiazolidine carboxylic acid (TCA) and thiazolidine carbonyl glycine (TCG)) and acetaldehyde (methyl thiazolidine carboxylic acid (MTCA) and methyl thiazolidine carbonyl glycine (MTCG)) was developed and validated for human urine and plasma samples. Targeting the sulfur-containing metabolites of formaldehyde and acetaldehyde in contrast to the commonly used biomarkers formate and acetate overcomes the high intra- and inter-individual variance. Due to their involvement in various endogenous processes, formate and acetate lack the required specificity for assessing the exposure to formaldehyde and acetaldehyde, respectively. Validation was successfully performed according to FDA's Guideline for Bioanalytical Method Validation (2018), showing excellent performance with regard to accuracy, precision, and limits of quantification (LLOQ). TCA, TCG, and MTCG proved to be stable under all investigated conditions, whereas MTCA showed a depletion after 21 months. The method was applied to a set of pilot samples derived from smokers who consumed unfiltered cigarettes spiked with 13C-labeled propylene glycol and 13C-labeled glycerol. These compounds were used as potential precursors for the formation of 13C-formaldehyde and 13C-acetaldehyde during combustion. Plasma concentrations were significantly lower as compared to urine, suggesting urine as suitable matrix for a biomonitoring. A smoking-related increase of unlabeled biomarker concentrations could not be shown due to the ubiquitous distribution in the environment. While the metabolites of 13C-acetaldehyde were not detected, the described method allowed for the quantification of 13C-formaldehyde uptake from cigarette smoking by targeting the biomarkers 13C-TCA and 13C-TCG in urine.Graphical abstract.


Assuntos
Acetaldeído/metabolismo , Formaldeído/metabolismo , Enxofre/sangue , Enxofre/urina , Acetaldeído/efeitos adversos , Biomarcadores/sangue , Biomarcadores/metabolismo , Biomarcadores/urina , Formaldeído/efeitos adversos , Glicina/análogos & derivados , Glicina/metabolismo , Humanos , Limite de Detecção , Metilação , Prolina/análogos & derivados , Prolina/sangue , Prolina/metabolismo , Prolina/urina , Fumar/efeitos adversos , Fumar/sangue , Fumar/metabolismo , Fumar/urina , Enxofre/metabolismo , Espectrometria de Massas em Tandem/métodos , Tiazolidinas/sangue , Tiazolidinas/metabolismo , Tiazolidinas/urina
5.
Chem Res Toxicol ; 33(7): 1989-1996, 2020 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-32633961

RESUMO

Formaldehyde (FA) is a human carcinogen that is ubiquitous in the ambient environment and also generated endogenously in oxidatively stressed cells. There is accumulated evidence that FA is an etiological agent of leukemia development in humans. To develop a biomarker for FA exposure, we have, in this study, developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) coupled with stable isotope-dilution method to explore the reactivity of FA with glutathione (GSH) in physiologically relevant conditions. Interestingly, analysis of the reaction mixture by LC-MS/MS revealed exposure concentration- and duration-dependent formation of thioproline-glycine (SPro-Gly) produced by reaction of FA with cysteinyl-glycine (Cys-Gly) as a novel metabolite. Furthermore, dose-dependent formation of the thioproline adduct was observed in human cells separately exposed to FA and Fe2+-EDTA, a hydroxyl radical source. To the best of our knowledge, this is the first study reporting a thiazolidine carboxylic acid formed by reaction of FA and Cys-Gly is a major metabolite of FA. The results suggest a variety of GSH-derived thiazolidine metabolites may serve as potential biomarkers for FA and oxidative stress exposure, and the developed LC-MS/MS method provides a means for accurate determination of SPro-Gly as a dosimeter of oxidative stress and formaldehyde exposure.


Assuntos
Formaldeído/toxicidade , Glutationa/farmacologia , Glicina/metabolismo , Estresse Oxidativo , Tiazolidinas/metabolismo , Biomarcadores/metabolismo , Cromatografia Líquida , Cisteína/química , Formaldeído/química , Glutationa/química , Glicina/química , Células Hep G2 , Humanos , Técnicas de Diluição do Indicador , Espectrometria de Massas em Tandem , Tiazolidinas/química
6.
Int J Mol Sci ; 21(10)2020 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-32443403

RESUMO

It is well-established that aminothiols, to which cysteine (Cys) belongs, are highly reactive towards aldehydes in an aqueous environment, forming substituted thiazolidine carboxylic acids. This report provides evidence that formation of the product containing a thiazolidine ring through non-enzymatic condensation of Cys and an active form of vitamin B6 pyridoxal 5'-phosphate (PLP) occurs in vivo in humans. To prove this point, a new method, based on a gas chromatography coupled with mass spectrometry (GC-MS), has been designed to identify and quantify Cys and PLP adduct, 2-(3-hydroxy-5-phosphonooxymethyl-2-methyl-4-pyridyl)-1,3-thiazolidine-4-carboxylic acid (HPPTCA) in human plasma. The GC-MS assay relies on sample deproteinization by ultrafiltration over cut-off membranes and preconcentration by drying under vacuum, followed by treatment of the residue with derivatization mixture containing anhydrous pyridine, N-trimethylsilyl-N-methyl trifluoroacetamide (MSTFA) and trimethylchlorosilane (TMCS). The method quantifies HPPTCA in a linear range from 1 to 20 µmol L-1, where the lowest standard on the calibration curve refers to the limit of quantification (LOQ). The validity of the method was demonstrated. Furthermore, the method was successfully applied to plasma samples donated by apparently healthy volunteers and breast cancer patients. The GC-MS assay provides a new tool that will hopefully facilitate studies on the role of HPPTCA in living systems.


Assuntos
Cisteína/metabolismo , Plasma/metabolismo , Fosfato de Piridoxal/metabolismo , Tiazolidinas/sangue , Cromatografia Gasosa-Espectrometria de Massas , Humanos , Tiazolidinas/metabolismo
7.
Biochem J ; 477(9): 1745-1757, 2020 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-32301498

RESUMO

Formaldehyde (HCHO) is a reactive carbonyl compound that formylates and cross-links proteins, DNA, and small molecules. It is of specific concern as a toxic intermediate in the design of engineered pathways involving methanol oxidation or formate reduction. The interest in engineering these pathways is not, however, matched by engineering-relevant information on precisely why HCHO is toxic or on what damage-control mechanisms cells deploy to manage HCHO toxicity. The only well-defined mechanism for managing HCHO toxicity is formaldehyde dehydrogenase-mediated oxidation to formate, which is counterproductive if HCHO is a desired pathway intermediate. We therefore sought alternative HCHO damage-control mechanisms via comparative genomic analysis. This analysis associated homologs of the Escherichia coli pepP gene with HCHO-related one-carbon metabolism. Furthermore, deleting pepP increased the sensitivity of E. coli to supplied HCHO but not other carbonyl compounds. PepP is a proline aminopeptidase that cleaves peptides of the general formula X-Pro-Y, yielding X + Pro-Y. HCHO is known to react spontaneously with cysteine to form the close proline analog thioproline (thiazolidine-4-carboxylate), which is incorporated into proteins and hence into proteolytic peptides. We therefore hypothesized that certain thioproline-containing peptides are toxic and that PepP cleaves these aberrant peptides. Supporting this hypothesis, PepP cleaved the model peptide Ala-thioproline-Ala as efficiently as Ala-Pro-Ala in vitro and in vivo, and deleting pepP increased sensitivity to supplied thioproline. Our data thus (i) provide biochemical genetic evidence that thioproline formation contributes substantially to HCHO toxicity and (ii) make PepP a candidate damage-control enzyme for engineered pathways having HCHO as an intermediate.


Assuntos
Endopeptidases , Escherichia coli , Formaldeído/metabolismo , Prolina/metabolismo , Aldeído Oxirredutases/metabolismo , Proteínas de Bactérias/metabolismo , Cisteína/metabolismo , Endopeptidases/genética , Endopeptidases/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Formaldeído/toxicidade , Genes Bacterianos , Genoma Bacteriano , Tiazolidinas/metabolismo
8.
J Med Chem ; 63(9): 4880-4895, 2020 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-32298120

RESUMO

Due to their role in many important signaling pathways, phosphatidylinositol 5-phosphate 4-kinases (PI5P4Ks) are attractive targets for the development of experimental therapeutics for cancer, metabolic, and immunological disorders. Recent efforts to develop small molecule inhibitors for these lipid kinases resulted in compounds with low- to sub-micromolar potencies. Here, we report the identification of CVM-05-002 using a high-throughput screen of PI5P4Kα against our in-house kinase inhibitor library. CVM-05-002 is a potent and selective inhibitor of PI5P4Ks, and a 1.7 Å X-ray structure reveals its binding interactions in the ATP-binding pocket. Further investigation of the structure-activity relationship led to the development of compound 13, replacing the rhodanine-like moiety present in CVM-05-002 with an indole, a potent pan-PI5P4K inhibitor with excellent kinome-wide selectivity. Finally, we employed isothermal cellular thermal shift assays (CETSAs) to demonstrate the effective cellular target engagement of PI5P4Kα and -ß by the inhibitors in HEK 293T cells.


Assuntos
Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Piridinas/farmacologia , Sulfonamidas/farmacologia , Tiazolidinas/farmacologia , Cristalografia por Raios X , Descoberta de Drogas , Células HEK293 , Ensaios de Triagem em Larga Escala , Humanos , Estrutura Molecular , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Ligação Proteica , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/metabolismo , Piridinas/síntese química , Piridinas/metabolismo , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Relação Estrutura-Atividade , Sulfonamidas/síntese química , Sulfonamidas/metabolismo , Tiazolidinas/síntese química , Tiazolidinas/metabolismo
9.
Pak J Pharm Sci ; 33(2): 575-579, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32276900

RESUMO

We report here the synthesis as well as antioxidant activity of a series of 2-aryl thiazolidine-4-carboxylic acids, including two novel derivatives. They were synthesized by nucleophilic cyclic condensation of L-cysteine hydrochloride with a range of aromatic aldehydes. Their in vitro antioxidant activity was evaluated by DPPH radical scavenging assay. It was observed that the aromatic substituent at C-2 of thiazolidine ring effects the antioxidant potential of the thiazolidine derivatives. The nature and position of the substituents on aromatic ring were correlated with antioxidant activity. Compounds with -OCH3 group on aromatic ring showed a better radical scavenging property than the other groups such as -Cl, -F, and -NO2. The presence of phenyl ring thus enhanced radical scavenging activity.


Assuntos
Antioxidantes/síntese química , Antioxidantes/metabolismo , Química Farmacêutica/métodos , Tiazolidinas/síntese química , Tiazolidinas/metabolismo , Avaliação Pré-Clínica de Medicamentos/métodos
10.
Mini Rev Med Chem ; 20(12): 1091-1100, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32107992

RESUMO

This article provides comprehensive and collective facts about teneligliptin. Teneligliptin is a dipeptide peptidase-4 (DPP-4) inhibitor that belongs to the third generation, used in the management of type 2 diabetes. It inhibits human DPP-4 enzyme activity. This drug falls under class 3; it interacts with S1, S2, and S2E extensive sub-sites. Teneligliptin and its metabolites are mainly determined in the human plasma matrix by hyphenated chromatographic methods. These developed methods could be foreseen for their clinical applications. Moreover, the stress degradation studies of Teneligliptin under different stress conditions provide an insight into degradation pathways and help in the elucidation of the structure of the degradation products by liquid mass spectroscopy. These methods are also used for routine quality control analysis of teneligliptin in pharmaceutical dosage forms.


Assuntos
Inibidores da Dipeptidil Peptidase IV/química , Pirazóis/química , Tiazolidinas/química , Glicemia/análise , Doenças Cardiovasculares/etiologia , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/patologia , Dipeptidil Peptidase 4/química , Dipeptidil Peptidase 4/metabolismo , Inibidores da Dipeptidil Peptidase IV/efeitos adversos , Inibidores da Dipeptidil Peptidase IV/metabolismo , Inibidores da Dipeptidil Peptidase IV/uso terapêutico , Interações Medicamentosas , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Meia-Vida , Humanos , Pirazóis/efeitos adversos , Pirazóis/metabolismo , Pirazóis/uso terapêutico , Tiazolidinas/efeitos adversos , Tiazolidinas/metabolismo , Tiazolidinas/uso terapêutico
11.
Plant Cell Physiol ; 61(3): 445-456, 2020 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-32030404

RESUMO

Plant growth and development relies on the accurate positioning of the cell plate between dividing cells during cytokinesis. The cell plate is synthetized by a specialized structure called the phragmoplast, which contains bipolar microtubules that polymerize to form a framework with the plus ends at or near the division site. This allows the transport of Golgi-derived vesicles toward the plus ends to form and expand the cell plate. Actin filaments play important roles in cell plate expansion and guidance in plant cytokinesis at the late phase, but whether they are involved at the early phase is unknown. To investigate this further, we disrupted the actin filaments in cell cycle-synchronized tobacco BY-2 cells with latrunculin B (LatB), an actin polymerization inhibitor. We observed the cells under a transmission electron microscope or a spinning-disk confocal laser scanning microscope. We found that disruption of actin filaments by LatB caused the membrane vesicles at the equatorial plane of the cell plate to be dispersed rather than form clusters as they did in the untreated cells. The midzone constriction of phragmoplast microtubules also was perturbed in LatB-treated cells. The live cell imaging and kymograph analysis showed that disruption of actin filaments also changed the accumulation timing of NACK1 kinesin, which plays a crucial role in cell plate expansion. This suggests that there are two functionally different types of microtubules in the phragmoplast. Together, our results show that actin filaments regulate phragmoplast microtubules at the initial phase of plant cytokinesis.


Assuntos
Citoesqueleto de Actina/metabolismo , Citocinese/fisiologia , Citoplasma/metabolismo , Microtúbulos/metabolismo , Compostos Bicíclicos Heterocíclicos com Pontes/metabolismo , Divisão Celular , Cinesina/metabolismo , Desenvolvimento Vegetal/fisiologia , Tiazolidinas/metabolismo , Tabaco/metabolismo
12.
Biomolecules ; 10(2)2020 02 10.
Artigo em Inglês | MEDLINE | ID: mdl-32050703

RESUMO

Microbial co-culture or mixed fermentation proved to be an efficient strategy to expand chemical diversity by the induction of cryptic biosynthetic pathways, and in many cases led to the production of new antimicrobial agents. In the current study, we report a rare example of the induction of silent/cryptic bacterial biosynthetic pathway by the co-culture of Durum wheat plant roots-associated bacterium Pantoea aggolomerans and date palm leaves-derived fungus Penicillium citrinum. The initial co-culture indicated a clear fungal growth inhibition which was confirmed by the promising antifungal activity of the co-culture total extract against Pc. LC-HRMS chemical profiling demonstrated a huge suppression in the production of secondary metabolites (SMs) of axenic cultures of both species with the emergence of new metabolites which were dereplicated as a series of siderophores. Large-scale co-culture fermentation led to the isolation of two new pulicatin derivatives together with six known metabolites which were characterised using HRESIMS and NMR analyses. During the in vitro antimicrobial evaluation of the isolated compounds, pulicatin H (2) exhibited the strongest antifungal activity against Pc, followed by aeruginaldehyde (1) and pulicatin F (4), hence explaining the initial growth suppression of Pc in the co-culture environment.


Assuntos
Pantoea/química , Pantoea/metabolismo , Tiazolidinas/metabolismo , Antibacterianos , Anti-Infecciosos , Antifúngicos , Técnicas de Cocultura , Fermentação , Pantoea/fisiologia , Penicillium , Raízes de Plantas , Sideróforos
13.
J Proteomics ; 210: 103541, 2020 01 06.
Artigo em Inglês | MEDLINE | ID: mdl-31614210

RESUMO

Recently it was discovered that thioproline, an unnatural analog of proline, can arise in vivo from the reaction of cysteine and formaldehyde in cells under oxidative stress. Sequence-specific bioincorporation of thioproline into proteins was studied via shotgun proteomics of Escherichia coli (E. coli) cells. In a strain auxotrophic for proline, thioproline was found widely incorporated in lieu of proline when the cells were incubated with thioproline. In total 1428 proteins and 235 distinct thioproline-containing peptides were identified. Label-free relative quantitation revealed 102 differentially expressed proteins (82 up-regulated and 20 down-regulated) in the thioproline-treated group (with thioproline in the medium) relative to the control group (with proline in the culture medium). Pathway enrichment analysis of the differentially expressed proteins showed that amino acid biosynthesis and protein synthesis has been most affected by thioproline exposure, as expected. Phenotypically, the thioproline-treated group was found to exhibit slower cell growth and stronger antioxidant capacity relative to the control. SIGNIFICANCE: Thioproline is a secondary metabolite of formaldehyde and a structural analog of proline. It is also known to exhibit a wide variety of pharmaceutical properties, but its exact biochemical role in the cell has not been elucidated. In this paper, we studied thioproline misincorporation (in lieu of proline) events during protein synthesis in E. coli. Global proteome profiling revealed that thioproline is extensively misincorporated throughout the proteome in E. coli cells exposed to thioproline, and pathways related to amino acid and protein biosynthesis are up-regulated. In addition, we demonstrated that pretreatment with thioproline appeared to increase E. coli cells' capacity to tolerate oxidative stress. Our findings suggest a novel explanation of thioproline's known antioxidative properties. This is, to our knowledge, the first ever study of thioproline misincorporation at the proteome level in any organism.


Assuntos
Antioxidantes/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Estresse Oxidativo , Proteoma/metabolismo , Proteômica/métodos , Tiazolidinas/metabolismo , Aminoácidos/metabolismo , Escherichia coli/crescimento & desenvolvimento , Proteoma/análise , Tiazolidinas/química
14.
Mol Cell Biol ; 40(4)2020 01 30.
Artigo em Inglês | MEDLINE | ID: mdl-31791978

RESUMO

Cyclase-associated protein 1 (CAP1) is a conserved actin-regulating protein that enhances actin filament dynamics and also regulates adhesion in mammalian cells. We previously found that phosphorylation at the Ser307/Ser309 tandem site controls its association with cofilin and actin and is important for CAP1 to regulate the actin cytoskeleton. Here, we report that transient Ser307/Ser309 phosphorylation is required for CAP1 function in both actin filament disassembly and cell adhesion. Both the phosphomimetic and the nonphosphorylatable CAP1 mutant, which resist transition between phosphorylated and dephosphorylated forms, had defects in rescuing the reduced rate of actin filament disassembly in the CAP1 knockdown HeLa cells. The phosphorylation mutants also had defects in alleviating the elevated focal adhesion kinase (FAK) activity and the enhanced focal adhesions in the knockdown cells. In dissecting further phosphoregulatory cell signals for CAP1, we found that cyclin-dependent kinase 5 (CDK5) phosphorylates both Ser307 and Ser309 residues, whereas cAMP signaling induces dephosphorylation at the tandem site, through its effectors protein kinase A (PKA) and exchange proteins directly activated by cAMP (Epac). No evidence supports an involvement of activated protein phosphatase in executing the dephosphorylation downstream from cAMP, whereas preventing CAP1 from accessing its kinase CDK5 appears to underlie CAP1 dephosphorylation induced by cAMP. Therefore, this study provides direct cellular evidence that transient phosphorylation is required for CAP1 functions in both actin filament turnover and adhesion, and the novel mechanistic insights substantially extend our knowledge of the cell signals that function in concert to regulate CAP1 by facilitating its transient phosphorylation.


Assuntos
Citoesqueleto de Actina/metabolismo , Adesão Celular/fisiologia , Proteínas de Ciclo Celular/metabolismo , AMP Cíclico/metabolismo , Quinase 5 Dependente de Ciclina/metabolismo , Proteínas do Citoesqueleto/metabolismo , Fatores de Despolimerização de Actina/metabolismo , Actinas/metabolismo , Compostos Bicíclicos Heterocíclicos com Pontes/metabolismo , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Proteínas do Citoesqueleto/genética , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Células HEK293 , Células HeLa , Humanos , Proteínas dos Microfilamentos/metabolismo , Fosforilação , Transdução de Sinais/fisiologia , Tiazolidinas/metabolismo
15.
Biomed Chromatogr ; 34(2): e4721, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31656058

RESUMO

Teneligliptin is a recently developed dipeptidyl peptidase-4 (DPP-4) inhibitor for the treatment of type 2 diabetes mellitus. To study simultaneous pharmacokinetics of teneligliptin and its major active metabolite, teneligliptin sulfoxide in human plasma, we developed and validated a LC-MS/MS method. The analytes were detected in the positive mode using multiple reaction monitoring (teneligliptin: m/z 427.2→243.1; teneligliptin-d8 : m/z 435.2→251.3; teneligliptin sulfoxide: m/z 443.2→68.2). The method demonstrated accuracy, precision, and linearity over the concentration range of 5 to 1000 ng/mL for teneligliptin and 2.5 to 500 ng/mL for teneligliptin sulfoxide. The developed method is the first fully validated method capable of simultaneous determination of teneligliptin and its active metabolite, teneligliptin sulfoxide in plasma. The suitability of the method was successfully demonstrated in terms of quantification of teneligliptin and teneligliptin sulfoxide pharmacokinetics in plasma samples collected from healthy volunteers. The measurement of plasma metabolite/parent ratio of teneligliptin was feasible by this method.


Assuntos
Cromatografia Líquida/métodos , Pirazóis/sangue , Espectrometria de Massas em Tandem/métodos , Tiazolidinas/sangue , Estabilidade de Medicamentos , Humanos , Limite de Detecção , Modelos Lineares , Pirazóis/química , Pirazóis/metabolismo , Pirazóis/farmacocinética , Reprodutibilidade dos Testes , Sulfóxidos/sangue , Sulfóxidos/química , Sulfóxidos/metabolismo , Sulfóxidos/farmacocinética , Tiazolidinas/química , Tiazolidinas/metabolismo , Tiazolidinas/farmacocinética
16.
Neurochem Res ; 45(2): 241-253, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31845170

RESUMO

This work evaluated the in vitro effect of thiazolidin-4-ones on the activity of AChE (total and isoforms) isolated from the cerebral cortex, hippocampus, and lymphocytes. Kinetic parameters were evaluated and molecular docking was performed. Our results showed that thiazolidinones derived from 4-(methylthio)benzaldehyde (1) and from 4-(methylsulfonyl)benzaldehyde (2) were capable of inhibiting the AChE activity in vitro. Three compounds, two with a propylpiperidine (1b and 2b) moiety and one with a 3-(diethylamino)propyl (1c) moiety showed IC50 values of 13.81 µM, and 3.13 µM (1b), 55.36 µM and 44.33 µM (1c) for cerebral cortex and hippocampus, respectively, and 3.11 µM for both (2b). Enzyme kinetics revealed that the type of AChE inhibition was mixed. Compound 1b inhibited the G1 and G4 AChE isoforms, while compounds 1c and 2b selectively inhibited the G4 isoform. Molecular docking showed a possible three-dimensional fit into the enzyme. Our findings showed that these thiazolidin-4-ones, especially those containing the propylpiperidine core, have a potential cholinesterase inhibitory activity and can be considered good candidates for future Alzheimer's therapy.


Assuntos
Acetilcolinesterase/metabolismo , Inibidores da Colinesterase/farmacologia , Tiazolidinas/farmacologia , Acetilcolinesterase/química , Animais , Domínio Catalítico , Inibidores da Colinesterase/síntese química , Inibidores da Colinesterase/metabolismo , Hipocampo/efeitos dos fármacos , Humanos , Isoenzimas/química , Isoenzimas/metabolismo , Cinética , Linfócitos/efeitos dos fármacos , Masculino , Simulação de Acoplamento Molecular , Estrutura Molecular , Ligação Proteica , Ratos Wistar , Tiazolidinas/síntese química , Tiazolidinas/metabolismo
17.
Biochim Biophys Acta Mol Cell Res ; 1866(10): 1634-1649, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31255721

RESUMO

Ligand-induced activation of Exchange Protein Activated by cAMP-1 (EPAC1) is implicated in numerous physiological and pathological processes, including cardiac fibrosis where changes in EPAC1 expression have been detected. However, little is known about how EPAC1 expression is regulated. Therefore, we investigated regulation of EPAC1 expression by cAMP in cardiac fibroblasts. Elevation of cAMP using forskolin, cAMP-analogues or adenosine A2B-receptor activation significantly reduced EPAC1 mRNA and protein levels and inhibited formation of F-actin stress fibres. Inhibition of actin polymerisation with cytochalasin-D, latrunculin-B or the ROCK inhibitor, Y-27632, mimicked effects of cAMP on EPAC1 mRNA and protein levels. Elevated cAMP also inhibited activity of an EPAC1 promoter-reporter gene, which contained a consensus binding element for TEAD, which is a target for inhibition by cAMP. Inhibition of TEAD activity using siRNA-silencing of its co-factors YAP and TAZ, expression of dominant-negative TEAD or treatment with YAP-TEAD inhibitors, significantly inhibited EPAC1 expression. However, whereas expression of constitutively-active YAP completely reversed forskolin inhibition of EPAC1-promoter activity it did not rescue EPAC1 mRNA levels. Chromatin-immunoprecipitation detected a significant reduction in histone3-lysine27-acetylation at the EPAC1 proximal promoter in response to forskolin stimulation. HDAC1/3 inhibition partially reversed forskolin inhibition of EPAC1 expression, which was completely rescued by simultaneously expressing constitutively active YAP. Taken together, these data demonstrate that cAMP downregulates EPAC1 gene expression via disrupting the actin cytoskeleton, which inhibits YAP/TAZ-TEAD activity in concert with HDAC-mediated histone deacetylation at the EPAC1 proximal promoter. This represents a novel negative feedback mechanism controlling EPAC1 levels in response to cAMP elevation.


Assuntos
AMP Cíclico/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Processamento de Proteína Pós-Traducional , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Amidas , Animais , Compostos Bicíclicos Heterocíclicos com Pontes/metabolismo , Técnicas de Cultura de Células , Linhagem Celular , Citocalasina D/metabolismo , Fibroblastos/metabolismo , Fatores de Troca do Nucleotídeo Guanina/genética , Histonas/metabolismo , Humanos , Masculino , Piridinas , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Tiazolidinas/metabolismo
18.
Comput Biol Chem ; 80: 512-523, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31185422

RESUMO

A new series of N'-(substituted phenyl)-5-chloro/iodo-3-phenyl-1H-indole-2-carbohydrazide (5, 6) and N-[2-(substituted phenyl)-4-oxo-1,3-thiazolidin-3-yl]-5-iodo/chloro-3-phenyl-1H-indole-2-carboxamide (7, 8) derivatives were synthesized and evaluated for their anticancer properties. Compounds 5a and 6b, selected as prototypes by the National Cancer Institute for screening against the full panel of 60 human tumor cell lines at a minimum of five concentrations at 10-fold dilutions, demonstrated remarkable antiproliferative activity against leukemia, non-small cell lung cancer, colon cancer, central nervous system (CNS) cancer, melanoma, ovarian cancer, renal cancer, and breast cancer (MCF-7) cell lines with GI50 values < 0.4 µM. A subset of the compounds was then tested for their potential to inhibit tubulin polymerization. Compounds 6f and 6g showed significant cytotoxicity at the nM level on MCF-7 cells and exhibited significant inhibitory activity on tubulin assembly and colchicine binding at about the same level as combretastatin A-4. Finally, docking calculations were performed to identify the binding mode of these compounds. Group 5 and 6 compounds interacted with the colchicine binding site through hydrophobic interactions similar to those of colchicine. These compounds with antiproliferative activity at high nanomolar concentration can serve as scaffolds for the design of novel microtubule targeting agents.


Assuntos
Antineoplásicos/farmacologia , Hidrazinas/farmacologia , Indóis/farmacologia , Tiazolidinas/farmacologia , Moduladores de Tubulina/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Antineoplásicos/metabolismo , Sítios de Ligação , Proliferação de Células/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Hidrazinas/síntese química , Hidrazinas/química , Hidrazinas/metabolismo , Indóis/síntese química , Indóis/química , Indóis/metabolismo , Células MCF-7 , Simulação de Acoplamento Molecular , Ligação Proteica , Tiazolidinas/síntese química , Tiazolidinas/química , Tiazolidinas/metabolismo , Moduladores de Tubulina/síntese química , Moduladores de Tubulina/química , Moduladores de Tubulina/metabolismo
19.
Nat Struct Mol Biol ; 26(7): 613-618, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31235915

RESUMO

Abasic (AP) sites are one of the most common DNA lesions that block replicative polymerases. 5-hydroxymethylcytosine binding, embryonic stem cell-specific protein (HMCES) recognizes and processes these lesions in the context of single-stranded DNA (ssDNA). A HMCES DNA-protein cross-link (DPC) intermediate is thought to shield the AP site from endonucleases and error-prone polymerases. The highly evolutionarily conserved SOS-response associated peptidase (SRAP) domain of HMCES and its Escherichia coli ortholog YedK mediate lesion recognition. Here we uncover the basis of AP site protection by SRAP domains from a crystal structure of the YedK DPC. YedK forms a stable thiazolidine linkage between a ring-opened AP site and the α-amino and sulfhydryl substituents of its amino-terminal cysteine residue. The thiazolidine linkage explains the remarkable stability of the HMCES DPC, its resistance to strand cleavage and the proteolysis requirement for resolution. Furthermore, its structure reveals that HMCES has specificity for AP sites in ssDNA at junctions found when replicative polymerases encounter the AP lesion.


Assuntos
DNA de Cadeia Simples/química , Proteínas de Ligação a DNA/química , Tiazolidinas/química , Cristalografia por Raios X , Reparo do DNA , Replicação do DNA , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/química , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Humanos , Simulação de Acoplamento Molecular , Conformação Proteica , Tiazolidinas/metabolismo
20.
Cell Mol Life Sci ; 76(17): 3349-3361, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31073744

RESUMO

The actin-related protein complex 2/3 (Arp2/3) generates branched actin networks important for many cellular processes such as motility, vesicular trafficking, cytokinesis, and intercellular junction formation and stabilization. Activation of Arp2/3 requires interaction with actin nucleation-promoting factors (NPFs). Regulation of Arp2/3 activity is achieved by endogenous inhibitory proteins through direct binding to Arp2/3 and competition with NPFs or by binding to Arp2/3-induced actin filaments and disassembly of branched actin networks. Arp2/3 inhibition has recently garnered more attention as it has been associated with attenuation of cancer progression, neurotoxic effects during drug abuse, and pathogen invasion of host cells. In this review, we summarize current knowledge on expression, inhibitory mechanisms and function of endogenous proteins able to inhibit Arp2/3 such as coronins, GMFs, PICK1, gadkin, and arpin. Moreover, we discuss cellular consequences of pharmacological Arp2/3 inhibition.


Assuntos
Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , 4-Butirolactona/análogos & derivados , 4-Butirolactona/química , 4-Butirolactona/metabolismo , Citoesqueleto de Actina , Complexo 2-3 de Proteínas Relacionadas à Actina/antagonistas & inibidores , Ligação Competitiva , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Endossomos/metabolismo , Fator de Maturação da Glia/química , Fator de Maturação da Glia/metabolismo , Humanos , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Tiazolidinas/química , Tiazolidinas/metabolismo
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