RESUMEN
Topoisomerases are proven drug targets, but antibiotics that poison bacterial Topoisomerase 1 (Top1) have yet to be discovered. We have developed a rapid and direct assay for quantification of Top1-DNA adducts that is suitable for high throughput assays. Adducts are recovered by "RADAR fractionation", a quick, convenient approach in which cells are lysed in chaotropic salts and detergent and nucleic acids and covalently bound adducts then precipitated with alcohol. Here we show that RADAR fractionation followed by ELISA immunodetection can quantify adducts formed by wild-type and mutant Top1 derivatives encoded by two different bacterial pathogens, Y. pestis and M. tuberculosis, expressed in E. coli or M. smegmatis, respectively. For both enzymes, quantification of adducts by RADAR/ELISA produces results comparable to the more cumbersome classical approach of CsCl density gradient fractionation. The experiments reported here establish that RADAR/ELISA assay offers a simple way to characterize Top1 mutants and analyze kinetics of adduct formation and repair. They also provide a foundation for discovery and optimization of drugs that poison bacterial Top1 using standard high-throughput approaches.
Asunto(s)
Proteínas Bacterianas/análisis , Fraccionamiento Celular/métodos , Aductos de ADN/análisis , ADN-Topoisomerasas de Tipo I/análisis , Ensayo de Inmunoadsorción Enzimática/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/aislamiento & purificación , Aductos de ADN/aislamiento & purificación , ADN-Topoisomerasas de Tipo I/aislamiento & purificación , Escherichia coli/química , Escherichia coli/genética , Escherichia coli/metabolismo , Immunoblotting/métodos , Mycobacterium smegmatis/química , Mycobacterium smegmatis/genética , Mycobacterium smegmatis/metabolismo , Reproducibilidad de los Resultados , Yersinia pestis/genéticaRESUMEN
Activation of efflux systems and the formation of biofilm are majorly adapted by microbes to resist antimicrobial agents. PPEF (bisbenzimidazole) targeting topoisomerase IA is observed to be an effective bactericidal agent against both Gram-positive and Gram-negative bacterial strains and thus can be developed as potent broad-spectrum antibiotic against MDR strains. PPEF treatment did not cause target specific mutation instead it leads to up-regulation of efflux gene in E. coli K12 as a mechanism of resistance. Microscopy, fluorescence spectroscopy and flow cytometry result demonstrate higher accumulation of PPEF in efflux gene deleted E. coli K12 mutants, and also suggest that Carbonyl Cyanide 3-Chlorophenylhydrazone (CCCP), resist the efflux of PPEF, and thus increases efficacy of PPEF. Herein, we report, PPEF and CCCP synergistically killed the persistent bacterial cells, which are not killed by PPEF alone. The above two compounds together inhibited biofilm formation, eradicate preformed biofilms and kills the biofilm cells of P. aeruginosa. PPEF and CCCP together reduced bacterial load of E. coli ATCC25922 by 6 log10 in neutropenic thigh infection model of balb/c mice. Present study suggests that combination therapy could be a promising antimicrobial strategy to handle MDR pathogenic strains.
Asunto(s)
Antibacterianos/farmacología , Biopelículas/efectos de los fármacos , Bisbenzimidazol/farmacología , Farmacorresistencia Bacteriana Múltiple/efectos de los fármacos , Infecciones por Escherichia coli/tratamiento farmacológico , Hidrazonas/farmacología , Animales , Biopelículas/crecimiento & desarrollo , ADN-Topoisomerasas de Tipo I/genética , ADN-Topoisomerasas de Tipo I/metabolismo , Modelos Animales de Enfermedad , Combinación de Medicamentos , Farmacorresistencia Bacteriana Múltiple/genética , Sinergismo Farmacológico , Infecciones por Escherichia coli/microbiología , Infecciones por Escherichia coli/patología , Escherichia coli K12/efectos de los fármacos , Escherichia coli K12/genética , Escherichia coli K12/crecimiento & desarrollo , Escherichia coli K12/metabolismo , Femenino , Expresión Génica , Genes MDR/efectos de los fármacos , Ratones , Ratones Endogámicos BALB C , Pruebas de Sensibilidad Microbiana , Neutropenia/tratamiento farmacológico , Neutropenia/microbiología , Neutropenia/patología , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/crecimiento & desarrollo , Pseudomonas aeruginosa/metabolismo , Muslo/microbiología , Muslo/patologíaRESUMEN
Human telomeric G-quadruplex DNA stabilization has emerged as an exciting novel approach for anticancer drug development. In the present study, we have designed and synthesized three C2-symmetric bisubstituted bisbenzimidazole naphthalenediimide (NDI) ligands, ALI-C3 , BBZ-ARO, and BBZ-AROCH2 , which stabilize human telomeric G-quadruplex DNA with high affinity. Herein, we have studied the binding affinities and thermodynamic contributions of each of these molecules with G-quadruplex DNA and compared the same to those of the parent NDI analogue, BMSG-SH-3. Results of fluorescence resonance energy transfer and surface plasmon resonance demonstrate that these ligands have a higher affinity for G4-DNA over duplex DNA and induce the formation of a G-quadruplex. The binding equilibrium constants obtained from the microcalorimetry studies of BBZ-ARO, ALI-C3 , and BBZ-AROCH2 were 8.47, 6.35, and 3.41 µM, respectively, with h-telo 22-mer quadruplex. These showed 10 and 100 times lower binding affinity with h-telo 12-mer and duplex DNA quadruplexes, respectively. Analysis of the thermodynamic parameters obtained from the microcalorimetry study suggests that interactions were most favorable for BBZ-ARO among all of the synthesized compounds. The ΔGfree obtained from molecular mechanics Poisson-Boltzmann surface area calculations of molecular dynamics (MD) simulation studies suggest that BBZ-ARO interacted strongly with G4-DNA. MD simulation results showed the highest hydrogen bond occupancy and van der Waals interactions were between the side chains of BBZ-ARO and the DNA grooves. A significant inhibition of telomerase activity (IC50 = 4.56 µM) and induced apoptosis in cancer cell lines by BBZ-ARO suggest that this molecule has the potential to be developed as an anticancer agent.
RESUMEN
In an attempt to find potential anticancer agents, a series of novel ethyl 4-(3-(aryl)-1-phenyl-1H-pyrazol-4-yl)-2-oxo-6-(pyridin-3-yl)cyclohex-3-enecarboxylates 5a-i and 5-(3-(4-fluorophenyl)-1-phenyl-1H-pyrazol-4-yl)-3-(pyridin-3-yl)-4,5-dihydropyrazole-1-carbothioamides 6a-i were designed, synthesized and evaluated for their topoisomerase IIα inhibitory activity and in vitro cytotoxicity against a panel of cancerous cell lines (MCF-7, NCI-H460, HeLa) and a normal cell line (HEK-293T). Molecular docking studies of all the synthesized compounds into the binding site of topoisomerase IIα protein (PDB ID: 1ZXM) were performed to gain a comprehensive understanding into plausible binding modes. These compounds were also screened for in silico drug-likeliness properties on the basis of the absorption, distribution, metabolism and excretion (ADME) prediction. Among all the synthesized compounds, analogue 5d showed superior cytotoxicity with an IC50 value of 7.01±0.60µM for HeLa, 8.55±0.35µM for NCI-H460 and 14.31±0.90 for MCF-7 cancer cell lines. Further, compound 5d showed 70.82% inhibition of topoisomerase IIα at a concentration of 100µM with maximum docking score of -8.24. Results of ADME prediction revealed that most of these compounds showed in silico drug-likeliness properties within the ideal range.
Asunto(s)
Antineoplásicos/farmacología , Proteínas de Unión al ADN/antagonistas & inhibidores , Diseño de Fármacos , Simulación del Acoplamiento Molecular , Pirazoles/farmacología , Inhibidores de Topoisomerasa II/farmacología , Antígenos de Neoplasias/metabolismo , Antineoplásicos/síntesis química , Antineoplásicos/química , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , ADN-Topoisomerasas de Tipo II/metabolismo , Proteínas de Unión al ADN/metabolismo , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Estructura Molecular , Pirazoles/síntesis química , Pirazoles/química , Relación Estructura-Actividad , Inhibidores de Topoisomerasa II/síntesis química , Inhibidores de Topoisomerasa II/químicaRESUMEN
Novel bisbenzimidazole inhibitors of bacterial type IA topoisomerase are of interest for the development of new antibacterial agents that are impacted by target-mediated cross resistance with fluoroquinolones. The present study demonstrates the successful synthesis and evaluation of bisbenzimidazole analogues as Escherichia coli topoisomerase IA inhibitors. 5-(4-Propylpiperazin-1-yl)-2-[2'-(4-ethoxyphenyl)-5'-benzimidazolyl]benzimidazole (12b) showed significant relaxation inhibition activity against EcTopo 1A (IC50 = 2 ± 0.005 µM) and a tendency to chelate metal ion. Interestingly, these compounds did not show significant inhibition of E. coli DNA gyrase and hTop 1 even up to 100 µM. Compound 12b has shown lowest MIC against E. coli strains among 24 compounds evaluated. The binding affinity constant and binding free energy of 12b with EcTopo 1A was observed 6.8 × 10(6) M(-1) and -10.84 kcal mol(-1) from isothermal titration calorimetry (ITC), respectively. In vivo mouse systemic infection and neutropenic thigh model experimental results confirmed the therapeutic efficacy of 12b, suggesting further development of this class of compounds as antibacterial agents.
Asunto(s)
Antibacterianos/síntesis química , Bencimidazoles/síntesis química , ADN-Topoisomerasas de Tipo I/metabolismo , Escherichia coli/efectos de los fármacos , Piperazinas/síntesis química , Inhibidores de Topoisomerasa I/síntesis química , Animales , Antibacterianos/química , Antibacterianos/farmacología , Bencimidazoles/química , Bencimidazoles/farmacología , Girasa de ADN/química , Farmacorresistencia Bacteriana , Escherichia coli/enzimología , Escherichia coli/aislamiento & purificación , Infecciones por Escherichia coli/complicaciones , Infecciones por Escherichia coli/tratamiento farmacológico , Femenino , Ratones Endogámicos BALB C , Pruebas de Sensibilidad Microbiana , Simulación del Acoplamiento Molecular , Neutropenia/complicaciones , Piperazinas/química , Piperazinas/farmacología , Unión Proteica , Sepsis/tratamiento farmacológico , Relación Estructura-Actividad , Termodinámica , Inhibidores de Topoisomerasa I/química , Inhibidores de Topoisomerasa I/farmacologíaRESUMEN
OBJECTIVES: Antibiotic resistance in bacterial pathogens is a serious clinical problem. Novel targets are needed to combat increasing drug resistance in Escherichia coli. Our objective is to demonstrate that 2-(3,4-dimethoxyphenyl)-5-[5-(4-methylpiperazin-1-yl)-1H-benzimidazol-2yl]-1H-benzimidazole (DMA) inhibits E. coli DNA topoisomerase I more strongly than human topoisomerase I. In addition, DMA is non-toxic to mammalian cells at antibiotic dosage level. METHODS: In the present study, we have established DMA as an antibacterial compound by determining MICs, post-antibiotic effects (PAEs) and MBCs for different standard as well as clinical strains of E. coli. We have described the differential catalytic inhibitory mechanism of bis-benzimidazole, DMA, for human and E. coli topoisomerase I and topoisomerase II by performing different assays, including relaxation assays, cleavage-religation assays, DNA unwinding assays, ethidium bromide displacement assays, decatenation assays and DNA gyrase supercoiling assays. RESULTS: DMA significantly inhibited bacterial growth at a very low concentration, but did not affect human cell viability at higher concentrations. Activity assays showed that it preferentially targeted E. coli topoisomerase I over human topoisomerase I, topoisomerase II and gyrase. Cleavage-religation assays confirmed DMA as a poison inhibitor of E. coli topoisomerase I. This study illuminates new properties of DMA, which may be further modified to develop an efficient topoisomerase inhibitor that is selective towards bacterial topoisomerase I. CONCLUSIONS: This is the first report of a bis-benzimidazole acting as an E. coli topoisomerase I inhibitor. DMA is a safe, non-cytotoxic molecule to human cells at concentrations that are needed for antibacterial activity.