RESUMO
Our previous research suggests an important regulatory role of CK2-mediated phosphorylation of enzymes involved in the thymidylate biosynthesis cycle, i.e., thymidylate synthase (TS), dihydrofolate reductase (DHFR), and serine hydroxymethyltransferase (SHMT). The aim of this study was to show whether silencing of the CK2α gene affects TS and DHFR expression in A-549 cells. Additionally, we attempted to identify the endogenous kinases that phosphorylate TS and DHFR in CCRF-CEM and A-549 cells. We used immunodetection, immunofluorescence/confocal analyses, reverse transcription-quantitative polymerase chain reaction (RT-qPCR), in-gel kinase assay, and mass spectrometry analysis. Our results demonstrate that silencing of the CK2α gene in lung adenocarcinoma cells significantly increases both TS and DHFR expression and affects their cellular distribution. Additionally, we show for the first time that both TS and DHFR are very likely phosphorylated by endogenous CK2 in two types of cancer cells, i.e., acute lymphoblastic leukaemia and lung adenocarcinoma. Moreover, our studies indicate that DHFR is phosphorylated intracellularly by CK2 to a greater extent in leukaemia cells than in lung adenocarcinoma cells. Interestingly, in-gel kinase assay results indicate that the CK2α' isoform was more active than the CK2α subunit. Our results confirm the previous studies concerning the physiological relevance of CK2-mediated phosphorylation of TS and DHFR.
Assuntos
Adenocarcinoma de Pulmão , Tetra-Hidrofolato Desidrogenase , Humanos , Fosforilação , Tetra-Hidrofolato Desidrogenase/química , Timidilato Sintase/metabolismoRESUMO
Over 30 compounds, including para-, meta-, and ortho-phenylenediboronic acids, ortho-substituted phenylboronic acids, benzenetriboronic acids, di- and triboronated thiophenes, and pyridine derivatives were investigated as potential ß-lactamase inhibitors. The highest activity against KPC-type carbapenemases was found for ortho-phenylenediboronic acid 3a, which at the concentration of 8/4 mg/L reduced carbapenems' MICs up to 16/8-fold, respectively. Checkerboard assays revealed strong synergy between carbapenems and 3a with the fractional inhibitory concentrations indices of 0.1-0.32. The nitrocefin hydrolysis test and the whole cell assay with E. coli DH5α transformant carrying blaKPC-3 proved KPC enzyme being its molecular target. para-Phenylenediboronic acids efficiently potentiated carbapenems against KPC-producers and ceftazidime against AmpC-producers, whereas meta-phenylenediboronic acids enhanced only ceftazidime activity against the latter ones. Finally, the statistical analysis confirmed that ortho-phenylenediboronic acids act synergistically with carbapenems significantly stronger than other groups. Since the obtained phenylenediboronic compounds are not toxic to MRC-5 human fibroblasts at the tested concentrations, they can be considered promising scaffolds for the future development of novel KPC/AmpC inhibitors. The complexation of KPC-2 with the most representative isomeric phenylenediboronic acids 1a, 2a, and 3a was modeled by quantum mechanics/molecular mechanics calculations. Compound 3a reached the most effective configuration enabling covalent binding to the catalytic Ser70 residue.
Assuntos
Antibacterianos , Ceftazidima , Humanos , Antibacterianos/química , Ceftazidima/farmacologia , Escherichia coli , beta-Lactamases/química , Proteínas de Bactérias/metabolismo , Inibidores de beta-Lactamases/farmacologia , Inibidores de beta-Lactamases/química , Carbapenêmicos/farmacologia , Testes de Sensibilidade Microbiana , Compostos Azabicíclicos/farmacologia , Combinação de MedicamentosRESUMO
Antifungal N-phenacyl derivatives of 4,6- and 5,6-dibromobenzimidazoles are interesting substrates in the synthesis of new antimycotics. Unfortunately, their application is limited by the low synthesis yields and time-consuming separation procedure. In this paper, we present the optimization of the synthesis conditions and purification methods of N-phenacyldibromobenzimidazoles. The reactions were carried out in various base solvent-systems including K2CO3, NaH, KOH, t-BuOK, MeONa, NaHCO3, Et3N, Cs2CO3, DBU, DIPEA, or DABCO as a base, and MeCN, DMF, THF, DMSO, or dioxane as a solvent. The progress of the reaction was monitored using HPLC analysis. The best results were reached when the reactions were carried out in an NaHCO3-MeCN system at reflux for 24 h. Additionally, the cytotoxic activity of the synthesized compounds against MCF-7 (breast adenocarcinoma), A-549 (lung adenocarcinoma), CCRF-CEM (acute lymphoblastic leukemia), and MRC-5 (normal lung fibroblasts) was evaluated. We observed that the studied cell lines differed in sensitivity to the tested compounds with MCF-7 cells being the most sensitive, while A-549 cells were the least sensitive. Moreover, the cytotoxicity of the tested derivatives towards CCRF-CEM cells increased with the number of chlorine or fluorine substituents. Furthermore, some of the active compounds, i.e., 2-(5,6-dibromo-1H-benzimidazol-1-yl)-1-(3,4-dichlorophenyl)ethanone (4f), 2-(4,6-dibromo-1H-benzimidazol-1-yl)-1-(2,4,6-trichlorophenyl)ethanone (5g), and 2-(4,6-dibromo-1H-benzimidazol-1-yl)-1-(2,4,6-trifluorophenyl)ethanone (5j) demonstrated pro-apoptotic properties against leukemic cells with derivative 5g being the most effective.
Assuntos
Antineoplásicos , Antineoplásicos/química , Linhagem Celular Tumoral , Humanos , Células MCF-7 , SolventesRESUMO
Currently available chemotherapeutic treatments for blood cancers (leukemia) usually have strong side effects. More selective, efficient, and less toxic anticancer agents are needed. We synthesized seven, new, optically pure (12aS)-1,3,4,12a-tetrahydropyrazino[2,1-c][1,4],12(2H,11H)-dione derivatives and examined their cytotoxicity towards eight cancer cell lines, including urinary bladder (TCC-SUP, UM-UC-3, KU-19-9), colon (LoVo), and breast (MCF-7, MDA-MB-231) cancer representatives, as well as two leukemic cell lines (MV-4-11, CCRF-CEM) and normal murine fibroblasts (Balb/3T3) as reference cell line. Three of the seven newly-obtained compounds ((12aS)-8-bromo-2-(3-phenylbenzoyl)-1,3,4,12a-tetrahydropyrazino[2,1-c][1,4],12(2H,11H)-dione, (12aS)-8,9-dimethoxy-2-(4-phenylbenzoyl)-1,3,4,12a-tetrahydropyrazino[2,1-c][1,4],12(2H,11H)-dione and (12aS)-8-nitro-2-(4-phenylbenzoyl)-1,3,4,12a-tetrahydropyrazino[2,1-c][1,4],12(2H,11H)-dione, showed enhanced activity and selectivity toward the leukemic MV-4-11â cell lines when compared to our previously reported compounds, with IC50 values in the range of 2.9-5.6â µM. Additionally, (12aS)-9-nitro-2-(4-phenylbenzoyl)-1,3,4,12a-tetrahydropyrazino[2,1-c][1,4],12(2H,11H)-dione exhibited a strong cytotoxic effect against the leukemic CCRF-CEM (IC50 =6.1â µM) and MV-4-11 (IC50 =11.0â µM) cell lines, a moderate cytotoxic effect toward other tumor lines (IC50 =31.8-55.0â µM) and very weak cytotoxic effect toward the Balb/3T3 reference cell lines. Selected compounds were further evaluated for their potential to induce apoptotic cell death in MV-4-11 cells by measuring caspase-3 activity. We also established the crystal structure of three products and investigated the effect of 22 derivatives of 1,3,4,12a-tetrahydropyrazino[2,1-c][1,4],12(2H,11H)-dione on the activity of the cancer-associated enzyme autotaxin. All compounds proved to be weak inhibitors of autotaxin, although some (R) and (S) enantiomers had Ki values of 10-19â µM. The obtained results showed that the tested compounds exhibited a selective antileukemic effect, which appeared not to be related directly to autotaxin. Molecular targets responsible for this effect remain to be identified. The newly obtained compounds can be used in the search for new, selective anticancer therapies.
Assuntos
Antineoplásicos/química , Benzodiazepinas/química , Desenho de Fármacos , Animais , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Benzodiazepinas/metabolismo , Benzodiazepinas/farmacologia , Sítios de Ligação , Domínio Catalítico , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Cristalografia por Raios X , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Camundongos , Conformação Molecular , Diester Fosfórico Hidrolases/química , Diester Fosfórico Hidrolases/metabolismo , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
Protein kinase CK2 has been considered as an attractive drug target for anti-cancer therapy. The synthesis of N-hydroxypropyl TBBi and 2MeTBBi derivatives as well as their respective esters was carried out by using chemoenzymatic methods. Concomitantly with kinetic studies toward recombinant CK2, the influence of the obtained compounds on the viability of two human breast carcinoma cell lines (MCF-7 and MDA-MB-231) was evaluated using MTT assay. Additionally, an intracellular inhibition of CK2 as well as an induction of apoptosis in the examined cells after the treatment with the most active compounds were studied by Western blot analysis, phase-contrast microscopy and flow cytometry method. The results of the MTT test revealed potent cytotoxic activities for most of the newly synthesized compounds (EC50 4.90 to 32.77 µM), corresponding to their solubility in biological media. We concluded that derivatives with the methyl group decrease the viability of both cell lines more efficiently than their non-methylated analogs. Furthermore, inhibition of CK2 in breast cancer cells treated with the tested compounds at the concentrations equal to their EC50 values correlates well with their lipophilicity since derivatives with higher values of logP are more potent intracellular inhibitors of CK2 with better proapoptotic properties than their parental hydroxyl compounds.
Assuntos
Antineoplásicos/farmacologia , Apoptose , Benzimidazóis/química , Neoplasias da Mama/tratamento farmacológico , Caseína Quinase II/antagonistas & inibidores , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/farmacologia , Antineoplásicos/síntese química , Neoplasias da Mama/enzimologia , Neoplasias da Mama/patologia , Proliferação de Células , Feminino , Humanos , Células MCF-7 , Relação Estrutura-AtividadeRESUMO
Phosphodiesterase 5 (PDE5) is one of the most extensively studied phosphodiesterases that is highly specific for cyclic-GMP hydrolysis. PDE5 became a target for drug development based on its efficacy for treatment of erectile dysfunction. In the present study, we synthesized four novel analogues of the phosphodiesterase type 5 (PDE5) inhibitor-tadalafil, which differs in (i) ligand flexibility (rigid structure of tadalafil vs. conformational flexibility of newly synthesized compounds), (ii) stereochemistry associated with applied amino acid building blocks, and (iii) substitution with bromine atom in the piperonyl moiety. For both the intermediate and final compounds as well as for the parent molecule, we have established the crystal structures and performed a detailed analysis of their structural features. The initial screening of the cytotoxic effect on 16 different human cancer and non-cancer derived cell lines revealed that in most cases, the parent compound exhibited a stronger cytotoxic effect than new derivatives, except for two cell lines: HEK 293T (derived from a normal embryonic kidney, that expresses a mutant version of SV40 large T antigen) and MCF7 (breast adenocarcinoma). Two independent studies on the inhibition of PDE5 activity, based on both pure enzyme assay and modulation of the release of nitric oxide from platelets under the influence of tadalafil and its analogues revealed that, unlike a reference compound that showed strong PDE5 inhibitory activity, the newly obtained compounds did not have a noticeable effect on PDE5 activity in the range of concentrations tested. Finally, we performed an investigation of the toxicological effect of synthesized compounds on Caenorhabditis elegans in the highest applied concentration of 6a,b and 7a,b (160 µM) and did not find any effect that would suggest disturbance to the life cycle of Caenorhabditis elegans. The lack of toxicity observed in Caenorhabditis elegans and enhanced, strengthened selectivity and activity toward the MCF7 cell line made 7a,b good leading structures for further structure activity optimization and makes 7a,b a reasonable starting point for the search of new, selective cytotoxic agents.
Assuntos
Caenorhabditis elegans/enzimologia , Nucleotídeo Cíclico Fosfodiesterase do Tipo 5/metabolismo , Inibidores da Fosfodiesterase 5 , Piperazinas , Tadalafila , Animais , Avaliação Pré-Clínica de Medicamentos , Células HEK293 , Humanos , Células MCF-7 , Inibidores da Fosfodiesterase 5/síntese química , Inibidores da Fosfodiesterase 5/química , Inibidores da Fosfodiesterase 5/farmacologia , Piperazinas/síntese química , Piperazinas/química , Piperazinas/farmacologia , Tadalafila/análogos & derivados , Tadalafila/síntese química , Tadalafila/química , Tadalafila/farmacologiaRESUMO
Three series of the ß-pyrimidine alanines, including willardiine - a naturally occurring amino acid, were prepared from the l-serine-derived sulfamidates. Compounds 3b, 4a and 4b demonstrated antiproliferative activity toward the studied cancer cell lines, albeit the effect of these compounds on human brain astrocytoma MOG-G-CCM cells was more significant than on human neuroblastoma SK-N-AS cells. The cytosine analog of willardiine, compound 4b, reduced viability of MOG-G-CCM cells with EC50 = 36 ± 2 µM, more effectively than AMPA antagonist GYKI 52466. Willardiine showed possible capability of affecting invasiveness of glioblastoma U251 MG cells with no effect on their viability and morphology. Compound 3d, the ethyl ester of willardiine, featured activity toward binding domain hHS1S2I of the GluR2 receptor. Docking analysis revealed that the location mode of compound 3d at the S1S2 domain of hGluR2 (PDB ID: 3R7X) might differ from that of willardiine.
Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Pirimidinas/química , Pirimidinas/farmacologia , beta-Alanina/análogos & derivados , beta-Alanina/farmacologia , Alanina/análogos & derivados , Alanina/síntese química , Alanina/química , Alanina/farmacologia , Antineoplásicos/síntese química , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Humanos , Modelos Moleculares , Pirimidinas/síntese química , Uracila/síntese química , Uracila/química , Uracila/farmacologia , beta-Alanina/síntese químicaRESUMO
BACKGROUND: The combination effect of 5-fluorouracil (5-FU) with either CX-4945 or a new inhibitor of protein kinase CK2, namely 14B (4,5,6,7-tetrabromo-1-(3-bromopropyl)-2-methyl-1H-benzimidazole), on the viability of MCF-7 and triple-negative MDA-MB-231 breast cancer cell lines was studied. METHODS: Combination index (CI) values were determined using an MTT-based assay and the Chou-Talalay model. The effect of the tested drug combinations on pro-apoptotic properties and cell cycle progression was examined using flow cytometry. The activation of FAK, p38 MAPK, and ERK1/2 kinases and the expression of selected pro-apoptotic markers in MDA-MB-231 cell line after the combined treatment were evaluated by the western blot method. Confocal microscopy was used to examine actin network in MDA-MB-231. RESULTS: Our results showed that a synergistic effect (CI < 1) occurred in MDA-MB-231 after treatment with both combinations of 5-FU with 14B or CX-4945, whereas the combination of 5-FU and 14B evoked an antagonistic effect in MCF-7. We conclude that the synergistic interactions (CI < 1) observed for both the combinations of 5-FU and 14B or CX-4945 in MDA-MB-231 correlated with an activation of p38 MAPK, inhibition of FAK, increased expression of apoptogenic markers, prolongation of S-phase of cell cycle, and destabilization of actin network. CONCLUSIONS: The obtained results support the recent observation that CK2 inhibitors can improve 5-FU-based anticancer therapy and FAK kinase can be an attractive molecular target in breast cancer therapy.
Assuntos
Neoplasias da Mama/metabolismo , Fluoruracila/farmacologia , Quinase 1 de Adesão Focal/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Neoplasias da Mama/tratamento farmacológico , Caseína Quinase II/antagonistas & inibidores , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Regulação para Baixo , Sinergismo Farmacológico , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Células MCF-7 , Naftiridinas/farmacologia , Fenazinas/farmacologiaRESUMO
Dihydrofolate reductase (DHFR) is a prominent molecular target in antitumor, antibacterial, antiprotozoan, and immunosuppressive chemotherapies, and CK2 protein kinase is an ubiquitous enzyme involved in many processes, such as tRNA and rRNA synthesis, apoptosis, cell cycle or oncogenic transformation. We show for the first time that CK2α subunit strongly interacted with and phosphorylated DHFR in vitro. Using quartz crystal microbalance with dissipation monitoring (QCM-D) we determined DHFR-CK2α binding kinetic parameters (Kd below 0.5⯵M, konâ¯=â¯10.31â¯×â¯104â¯M-1s-1 and koffâ¯=â¯1.40â¯×â¯10-3s-1) and calculated Gibbs free energy (-36.4â¯kJ/mol). In order to identify phosphorylation site(s) we used site-directed mutagenesis to obtain several DHFR mutants with predicted CK2-phosphorylable serine or threonine residues substituted with alanines. All enzyme forms were subjected to CK2α subunit catalytic activity and the results pointed to serine 168 as a phosphorylation site. Mass spectrometry analyses confirmed the presence of phosphoserine 168 and revealed additionally the presence of phosphoserine 145, although the latter phosphorylation was on a very low level.
Assuntos
Tetra-Hidrofolato Desidrogenase/metabolismo , Caseína Quinase II/química , Caseína Quinase II/metabolismo , Domínio Catalítico , Humanos , Cinética , Fosforilação , Ligação Proteica , Mapas de Interação de Proteínas , Especificidade por SubstratoRESUMO
The 1H-1,2,3-triazole-originated derivatives of willardiine were obtained by: (i) construction of the 1H-1,2,3-triazole ring in 1,3-dipolar cycloaddition of the uracil-derived azides and the carboxylate-bearing alkynes or α-acylphosphorus ylide, or (ii) N-alkylation of the uracil derivative with the 1H-1,2,3-triazole-4-carboxylate-derived mesylate. The latter method offered: (i) reproducible results, (ii) a significant reduction of amounts of auxiliary materials, (iii) reduction in wastes and (iv) reduction in a number of manual operations required for obtaining the reaction product. Compound 6a exhibited significant binding affinity to hHS1S2I ligand-binding domain of GluR2 receptor (EC50â¯=â¯2.90⯵M) and decreased viability of human astrocytoma MOG-G-CCM cells in higher extent than known AMPA antagonist GYKI 52466.
Assuntos
Antineoplásicos/farmacologia , Ácidos Carboxílicos/farmacologia , Triazóis/farmacologia , Uracila/análogos & derivados , Uracila/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Ácidos Carboxílicos/síntese química , Ácidos Carboxílicos/química , Linhagem Celular Tumoral , Reação de Cicloadição , Humanos , Simulação de Acoplamento Molecular , Domínios Proteicos , Receptores de AMPA/química , Receptores de AMPA/metabolismo , Triazóis/síntese química , Triazóis/química , Uracila/síntese químicaRESUMO
Protein kinase 2 (CK2), a member of the serine/threonine kinase family, has been established as a promising target in anticancer therapy. New derivatives of known CK2 inhibitors 4,5,6,7-tetrabromo-1H-benzimidazole (TBBi) and 4,5,6,7-tetrabromo-1H-benzotriazole (TBBt) bearing azide or substituted triazole groups were synthesized. Their influence on the activity of human recombinant CK2α and cytotoxicity against normal and cancer cell lines were evaluated. TBBi derivatives with triazole substituted with carboxyl substituent (7 and 10) exhibited the most potent inhibitory activity against CK2 with Ki value in the range of 1.96-0.91µM, respectively. New TBBi derivatives 2, 3, 5 and 9 have demonstrated the EC50, in the range of 12-25µM and 13-29µM respectively towards CCRF-CEM and MCF-7 cells. Derivatives TBBi decreased viability of cancer cells more efficiently than BALB cells and the biggest differences were observed for the azide substituted compounds 3 and 5. The effect of the most active compounds on the activity of eight off-target kinases was evaluated. Inhibitory efficiency of CK2-mediated p65 phosphorylation was demonstrated for the TBBi and compound 12.
Assuntos
Antineoplásicos/farmacologia , Benzimidazóis/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Triazóis/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Benzimidazóis/síntese química , Benzimidazóis/química , Caseína Quinase II/antagonistas & inibidores , Caseína Quinase II/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Estrutura Molecular , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Relação Estrutura-Atividade , Triazóis/síntese química , Triazóis/químicaRESUMO
Thymidylate synthase (TS) may undergo phosphorylation endogenously in mammalian cells, and as a recombinant protein expressed in bacterial cells, as indicated by the reaction of purified enzyme protein with Pro-Q® Diamond Phosphoprotein Gel Stain (PGS). With recombinant human, mouse, rat, Trichinella spiralis and Caenorhabditis elegans TSs, expressed in Escherichia coli, the phosphorylated, compared to non-phosphorylated recombinant enzyme forms, showed a decrease in Vmax(app), bound their cognate mRNA (only rat enzyme studied), and repressed translation of their own and several heterologous mRNAs (human, rat and mouse enzymes studied). However, attempts to determine the modification site(s), whether endogenously expressed in mammalian cells, or recombinant proteins, did not lead to unequivocal results. Comparative ESI-MS/analysis of IEF fractions of TS preparations from parental and FdUrd-resistant mouse leukemia L1210 cells, differing in sensitivity to inactivation by FdUMP, demonstrated phosphorylation of Ser(10) and Ser(16) in the resistant enzyme only, although PGS staining pointed to the modification of both L1210 TS proteins. The TS proteins phosphorylated in bacterial cells were shown by (31)P NMR to be modified only on histidine residues, like potassium phosphoramidate (KPA)-phosphorylated TS proteins. NanoLC-MS/MS, enabling the use of CID and ETD peptide fragmentation methods, identified several phosphohistidine residues, but certain phosphoserine and phosphothreonine residues were also implicated. Molecular dynamics studies, based on the mouse TS crystal structure, allowed one to assess potential of several phosphorylated histidine residues to affect catalytic activity, the effect being phosphorylation site dependent.
Assuntos
Timidilato Sintase/metabolismo , Animais , Linhagem Celular Tumoral , Camundongos , Fosforilação , CoelhosRESUMO
The efficient method for the synthesis of novel cell permeable inhibitors of protein kinase CK2 with anticancer and proapoptotic activity has been developed. A series of polybrominated benzimiadazole derivatives substituted by various cyanoalkyl groups have been synthesized. Cyanoethyl derivatives were obtained by Michael type addition of 4,5,6,7-tetrabromo-1H-benzimidazole (TBBi) and 4,5,6,7-tetrabromo-2-methyl-1H-benzimidazole to acrylonitrile, whilst cyanomethyl, cyanopropyl and cyanobutyl analogs by N-alkylation of 4,5,6,7-tetrabromo-1H-benzimidazole and 4,5,6,7-tetrabromo-2-methyl-1H-benzimidazole with appropriate cyanoalkyl halides. The inhibitory activity against protein kinase rhCK2α catalytic subunit and cytotoxicity against two human cancer cell lines: acute lymphocytic leukemia (CCRF-CEM) and breast (MCF-7) were evaluated for all newly synthesized compounds. Additionally, the proapoptotic activity toward leukemia cells and intracellular inhibition of CK2 for the most cytotoxic derivatives have been performed, demonstrating 4,5,6,7-tetrabromo-2-methyl-1H-benzimidazole as a new selective inhibitor of rhCK2 with twenty-fold better proapoptotic activity than parental compound (TBBi).
Assuntos
Antineoplásicos/síntese química , Apoptose/efeitos dos fármacos , Benzimidazóis/síntese química , Caseína Quinase II/antagonistas & inibidores , Linfócitos/efeitos dos fármacos , Inibidores de Proteínas Quinases/síntese química , Antineoplásicos/farmacologia , Benzimidazóis/farmacologia , Caseína Quinase II/genética , Caseína Quinase II/metabolismo , Linhagem Celular Tumoral , Permeabilidade da Membrana Celular , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Halogenação , Humanos , Linfócitos/enzimologia , Linfócitos/patologia , Células MCF-7 , Inibidores de Proteínas Quinases/farmacologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Relação Estrutura-AtividadeRESUMO
Benzosiloxaboroles are an emerging class of medicinal agents possessing promising antimicrobial activity. Herein, the expedient synthesis of two novel thiol-functionalized benzosiloxaboroles 1e and 2e is reported. The presence of the SH group allowed for diverse structural modifications involving the thiol-Michael addition, oxidation, as well as nucleophilic substitution giving rise to a series of 27 new benzosiloxaboroles containing various polar functional groups, e.g., carbonyl, ester, amide, imide, nitrile, sulfonyl and sulfonamide, and pendant heterocyclic rings. The activity of the obtained compounds against selected bacterial and yeast strains, including multidrug-resistant clinical strains, was investigated. Compounds 6, 12, 20 and 22-24 show high activity against Staphylococcus aureus, including both methicillin-sensitive (MSSA) and methicillin-resistant (MRSA) strains, with MIC values in the range of 1.56-12.5 µg mL-1, while their cytotoxicity is relatively low. The in vitro assay performed with 2-(phenylsulfonyl)ethylthio derivative 20 revealed that, in contrast to the majority of known antibacterial oxaboroles, the plausible mechanism of antibacterial action, involving inhibition of the leucyl-tRNA synthetase enzyme, is not responsible for the antibacterial activity. Structural bioinformatic analysis involving molecular dynamics simulations provided a possible explanation for this finding.
RESUMO
CK2 and PIM-1 are serine/threonine kinases involved in the regulation of many essential processes, such as proliferation, differentiation, and apoptosis. Inhibition of CK2 and PIM-1 kinase activity has been shown to significantly reduce the viability of cancer cells by inducing apoptosis. A series of novel amino alcohol derivatives of parental DMAT were designed and synthesized as potent dual CK2/PIM-1 inhibitors. Concomitantly with the inhibition studies toward recombinant CK2 and PIM-1, the influence of the obtained compounds on the viability of three human carcinoma cell lines, i.e., acute lymphoblastic leukemia (CCRF-CEM), human chronic myelogenous leukemia (K-562), and breast cancer (MCF-7), as well as non-cancerous cells (Vero), was evaluated using an MTT assay. Induction of apoptosis and cell cycle progression after treatment with the most active compound and a lead compound were studied by flow-cytometry-based assay. Additionally, autophagy induction in K-562 cells and intracellular inhibition of CK2 and PIM-1 in all the tested cell lines were evaluated by qualitative/quantitative fluorescence-based assay and Western blot method, respectively. Among the newly developed inhibitors, 1,1,1-trifluoro-3-[(4,5,6,7-tetrabromo-1H-benzimidazol-2-yl)amino]propan-2-ol demonstrates the highest selectivity and the most prominent proapoptotic properties towards the studied cancer cells, especially towards acute lymphoblastic leukemia, in addition to inducing autophagy in K-562 cells.
RESUMO
Highly purified preparations of thymidylate synthase, isolated from calf thymus, and L1210 parental and FdUrd-resistant cells, were found to be nitrated, as indicated by a specific reaction with anti-nitro-tyrosine antibodies, suggesting this modification to appear endogenously in normal and tumor tissues. Each human, mouse and Ceanorhabditis elegans recombinant TS preparation, incubated in vitro in the presence of NaHCO(3), NaNO(2) and H(2)O(2) at pH 7.5, underwent tyrosine nitration, leading to a V(max)(app) 2-fold lower following nitration of 1 (with human or C. elegans TS) or 2 (with mouse TS) tyrosine residues per monomer. Enzyme interactions with dUMP, meTHF or 5-fluoro-dUMP were not distinctly influenced. Nitration under the same conditions of model tripeptides of a general formula H(2)N-Gly-X-Gly-COOH (X = Phe, Tyr, Trp, Lys, Arg, His, Ser, Thr, Cys, Gly), monitored by NMR spectroscopy, showed formation of nitro-species only for H-Gly-Tyr-Gly-OH and H-Gly-Phe-Gly-OH peptides, the chemical shifts for nitrated H-Gly-Tyr-Gly-OH peptide being in a very good agreement with the strongest peak found in (15)N-(1)H HMBC spectrum of nitrated protein. MS analysis of nitrated human and C. elegans proteins revealed several thymidylate synthase-derived peptides containing nitro-tyrosine (at positions 33, 65, 135, 213, 230, 258 and 301 in the human enzyme) and oxidized cysteine (human protein Cys(210), with catalytically critical Cys(195) remaining apparently unmodified) residues.
Assuntos
Timidilato Sintase/metabolismo , Tirosina/metabolismo , Animais , Caenorhabditis elegans/enzimologia , Bovinos , Linhagem Celular Tumoral , Humanos , Camundongos , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Timidilato Sintase/química , Timidilato Sintase/isolamento & purificação , Timo/enzimologia , Tirosina/químicaRESUMO
Two isomeric benzosiloxaborole derivatives 3a and 5a bearing fluorine and 4,4-dimethyl-2-oxazolin-2-yl substituents attached to the aromatic rings were obtained. Both compounds were prone to hydrolytic cleavage of the oxazoline ring after initial protonation or methylation of the nitrogen atom. The derivative 3c featuring N-methylammoniumalkyl ester functionality was successfully subjected to N-sulfonylation and N-acylation reactions to give respective derivatives which demonstrates its potential for modular synthesis of structurally extended benzosiloxaboroles. Compound 5c bearing N-ammoniumalkyl ester underwent conversion to a unique macrocyclic dimer due to siloxaborole ring opening. Furthermore, an unexpected 4-electron reduction of the oxazoline ring occurred during an attempted synthesis of 5a. The reaction gave rise to an unprecedented 7-membered heterocyclic system 4a comprising a relatively stable B-O-B-O-Si linkage and stabilized by an intramolecular N-B coordination. It could be cleaved to derivative 4c bearing BOH and SiMe2OH groups which acts as a pseudo-diol as demonstrated by formation of an adduct with Tavaborole. Apart from the multinuclear NMR spectroscopy characterization, crystal structures of the obtained products were determined in many cases by X-ray diffraction. Investigation of biological activity of the obtained compounds revealed that derivatives 3e and 3f with pendant N-methyl arylsulfonamide groups exhibit high activity against Gram-positive cocci such as methicillin-sensitive Staphylococcus aureus ATCC 6538P, methicillin-resistant S. aureus (MRSA) ATCC 43300 as well as the MRSA clinical strains, with MIC values in the range of 3.12-6.25 mg L-1. These two compounds also showed activity against Enterococcus faecalis ATCC 29212 and Enterococcus faecium ATCC 6057 (with MICs of 25-50 mg L-1). The results of the antimicrobial activity and cytotoxicity studies indicate that 3e and 3f can be considered as potential antibacterial agents, especially against S. aureus MRSA.
RESUMO
Thymidylate synthase (TS), dihydrofolate reductase (DHFR), and serine hydroxymethyltransferase (SHMT) constitute the thymidylate synthesis cycle providing thymidylate for DNA synthesis and repair. Our previous studies indicated that TS and DHFR are the substrates of protein kinase CK2. This work has been aimed at the elucidation of the effect of CK2 activity on cell cycle progression, thymidylate synthesis enzyme expression and localization, and the role of CK2-mediated TS phosphorylation in in vitro di- and trimolecular complex formation. The results were obtained by means of western blot, confocal microscopy, flow cytometry, quantitative polymerase chain reaction (QPCR), quartz crystal microbalance with dissipation monitoring (QCM-D), and microthermophoresis (MST). Our research indicates that CK2 inhibition does not change the levels of the transcripts; however, it affects the protein levels of DHFR and TS in both tested cell lines, i.e., A549 and CCRF-CEM, and the level of SHMT1 in CCRF-CEM cells. Moreover, we show that CK2-mediated phosphorylation of TS enables the protein (pTS) interaction with SHMT1 and leads to the stability of the tri-complex containing SHMT1, DHFR, and pTS. Our results suggest an important regulatory role of CK2-mediated phosphorylation for inter- and intracellular protein level of enzymes involved in the thymidylate biosynthesis cycle.
RESUMO
A series of new polybrominated benzimidazoles and benzotriazoles has been synthesized and their influence on the activity of protein kinase CK2 was evaluated. It was revealed that the most active inhibitors are those with methyl or ethyl substituent at benzene ring, namely 5,6,7-tribromo-4-methyl-1H-benzotriazole (38, IC(50) 0.51 µM) and 5,6,7-tribromo-4-ethyl-1H-benzotriazole (40, IC(50) 0.16 µM). The derivatives with large aromatic or heterocyclic substituents connected to benzimidazole or benzotriazole scaffold appeared to be less potent inhibitors.
Assuntos
Caseína Quinase II/antagonistas & inibidores , Desenho de Fármacos , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/farmacologia , Benzimidazóis/química , Benzimidazóis/farmacologia , Caseína Quinase II/metabolismo , Humanos , Concentração Inibidora 50 , Inibidores de Proteínas Quinases/química , Triazóis/química , Triazóis/farmacologiaRESUMO
Formylation of ansa[4]-ferrocene, obtained through the ruthenium-catalysed olefin metathesis, yields two separable, planar chiral 1,3- and 1,2-ansa-ferrocene aldehydes. Single-crystal X-ray structure analysis reveals that both regioisomers crystallize with spontaneous resolution of the racemate in the chiral P212121 space group with one molecule in the asymmetric unit. The major 1,3-isomer was further transformed into a conjugate with 1,2,3-triazole and uracil using "click" chemistry as the key synthetic step. This inorganic-organic hybrid displays anticancer activity (MCF-7, A549, MDA-MB-231 cell lines) with EC50 values comparable to those for cisplatin.