RESUMEN
Cervical cancer is a major cause of gynecological related mortalities in developing countries. Cisplatin, a potent chemotherapeutic agent used for treating advanced cervical cancer exhibits side effects and resistance development. The current study was aimed to investigate the repurposing of l-menthol as a potential therapeutic drug against cervical cancer. L-menthol was predicted to be non-toxic with good pharmacokinetic properties based on SwissADME and pkCSM analysis. Subsequently, 543 and 1664 targets of l-menthol and cervical cancer were identified using STITCH, BATMAN-TCM, PharmMapper and CTD databases. STRING and Cytoscape analysis of the merged protein-protein interaction network revealed 107 core targets of l- menthol against cervical cancer. M-CODE identified highly connected clusters between the core targets which through KEGG analysis were found to be enriched in pathways related to apoptosis and adherence junctions. Molecular docking showed that l- menthol targeted E6, E6AP and E7 onco-proteins of HPV that interact and inactivate TP53 and Rb1 in cervical cancer, respectively. Molecular docking also showed good binding affinity of l-menthol toward proteins associated with apoptosis and migration. Molecular dynamics simulation confirmed stability of the docked complexes. In vitro analysis confirmed that l-menthol was cytotoxic towards cervical cancer CaSki cells and altered expression of TP53, Rb1, CDKN1A, E2F1, NFKB1, Akt-1, caspase-3, CDH1 and MMP-2 genes identified through network pharmacology approach. Schematic representation of the work flow depicting the potential of l-menthol to target cervical cancer.
Asunto(s)
Mentol , Neoplasias del Cuello Uterino , Femenino , Humanos , Mentol/farmacología , Simulación del Acoplamiento Molecular , Farmacología en Red , Neoplasias del Cuello Uterino/tratamiento farmacológicoRESUMEN
Acinetobacter baumannii is an opportunistic pathogen known for high morbidity and mortality. It causes life-threatening infections, such as ventilator-associated pneumonia (VAP), bacteremia, meningitis, wound and urinary tract infections (UTI). Increase in carbapenem resistance exhibited by A. baumannii has accentuated the need for novel targets for effective treatment. Despite the pronounced relevance of PPK2 as a pathogenicity determinant in several pathogens, it has not been explored as a drug target in A. baumannii. The present study was piloted to investigate the substrate binding by A. baumannii PPK2 (AbPPK2), a two-domain Class II polyphosphate kinase 2. A homology model of AbPPK2 was developed and validated for molecular docking of ATP and ADP in the predicted binding pocket. Further analysis of AbPPK2 revealed a set of common residues in the catalytic cleft interacting with ATP and ADP which would be useful for the screening of inhibitors against A. baumannii.
Asunto(s)
Acinetobacter baumannii , Adenosina Difosfato/farmacología , Adenosina Monofosfato/farmacología , Adenosina Trifosfato/farmacología , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Simulación del Acoplamiento Molecular , Polifosfatos/metabolismoRESUMEN
BACKGROUND: Xenorhabdus nematophila maintains species-specific mutual interaction with nematodes of Steinernema genus. Type II Toxin Antitoxin (TA) systems, the mazEF TA system controls stress and programmed cell death in bacteria. OBJECTIVE: This study elucidates the functional characterization of Xn-mazEF, a mazEF homolog in X. nematophila by computational and in vitro approaches. METHODS: 3D- structural models for Xn-MazE toxin and Xn-MazF antitoxin were generated, validated and characterized for protein - RNA interaction analysis. Further biological and cellular functions of Xn-MazF toxin were also predicted. Molecular dynamics simulations of 50ns for Xn- MazF toxin complexed with nucleic acid units (DU, RU, RC, and RU) were performed. The MazF toxin and complete MazEF operon were endogenously expressed and monitored for the killing of Escherichia coli host cells under arabinose induced tightly regulated system. RESULTS: Upon induction, E. coli expressing toxin showed rapid killing within four hours and attained up to 65% growth inhibition, while the expression of the entire operon did not show significant killing. The observation suggests that the Xn-mazEF TA system control transcriptional regulation in X. nematophila and helps to manage stress or cause toxicity leading to programmed death of cells. CONCLUSION: The study provides insights into structural and functional features of novel toxin, Xn- MazF and provides an initial inference on control of X. nematophila growth regulated by TA systems.
Asunto(s)
Proteínas Bacterianas/fisiología , Proteínas de Unión al ADN/metabolismo , Endorribonucleasas/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/fisiología , Sistemas Toxina-Antitoxina/fisiología , Apoptosis/fisiología , Proteínas Bacterianas/genética , Proteínas de Unión al ADN/genética , Endorribonucleasas/genética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Simulación de Dinámica Molecular , Operón/genética , Factores de Tiempo , Sistemas Toxina-Antitoxina/genética , XenorhabdusRESUMEN
Acinetobacter baumannii is clinically one of the most significant pathogens, especially in intensive care settings, because of its multidrug-resistance (MDR). Repurposing of high-affinity drugs is a faster and more plausible approach for combating the emergence of MDR and to tackle bacterial infections. This study was aimed to evaluate the approved drugs potentially inhibiting A. baumannii PPK1 (AbPPK1) mediated synthesis of polyphosphates (polyP). Based on virtual screening, molecular dynamic simulation, and CD spectroscopy for thermal stability, two stable ligands, etoposide and genistein, were found with promising contours for further investigation. Following in vitro inhibition of AbPPK1, the efficacy of selected drugs was further tested against virulence traits of A. baumannii. These drugs significantly reduced the biofilm formation, surface motility in A. baumannii and led to decreased survival under desiccation. In addition to inhibition of PPK1, both drugs increased the expression of polyP degrading enzyme, exopolyphosphatase (PPX), that might be responsible for the decrease in the total cellular polyP. Since polyP modulates the virulence factors in bacteria, destabilization of the polyP pool by these drugs seems particularly striking for their therapeutic applications against A. baumannii.
Asunto(s)
Acinetobacter baumannii/enzimología , Acinetobacter baumannii/metabolismo , Antibacterianos/farmacología , Fosfotransferasas (Aceptor del Grupo Fosfato)/efectos de los fármacos , Fosfotransferasas (Aceptor del Grupo Fosfato)/metabolismo , Factores de Virulencia/genética , Ácido Anhídrido Hidrolasas/metabolismo , Infecciones por Acinetobacter/microbiología , Acinetobacter baumannii/efectos de los fármacos , Acinetobacter baumannii/genética , Acil-Butirolactonas/metabolismo , Biopelículas/crecimiento & desarrollo , Clonación Molecular , Farmacorresistencia Bacteriana Múltiple/efectos de los fármacos , Regulación Bacteriana de la Expresión Génica , Pruebas de Sensibilidad Microbiana , Simulación del Acoplamiento Molecular , Fosfotransferasas (Aceptor del Grupo Fosfato)/química , Fosfotransferasas (Aceptor del Grupo Fosfato)/genética , Polifosfatos/metabolismo , Análisis de Secuencia , Virulencia/genéticaRESUMEN
Bacterial infections have always been an unrestrained challenge to the medical community due to the rise of multi-drug tolerant and resistant strains. Pioneering work on Escherichia coli polyphosphate kinase (PPK) by Arthur Kornberg has generated great interest in this polyphosphate (PolyP) synthesizing enzyme. PPK has wide distribution among pathogens and is involved in promoting pathogenesis, stress management and susceptibility to antibiotics. Further, the absence of a PPK orthologue in humans makes it a potential drug target. This review covers the functional and structural aspects of polyphosphate kinases in bacterial pathogens. A description of molecules being designed against PPKs has been provided, challenges associated with PPK inhibitor design are highlighted and the strategies to enable development of efficient drug against this enzyme have also been discussed.
Asunto(s)
Antibacterianos/farmacología , Bacterias/patogenicidad , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Animales , Antibacterianos/síntesis química , Antibacterianos/química , Bacterias/efectos de los fármacos , Bacterias/enzimología , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Diseño de Fármacos , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Humanos , Modelos Moleculares , Fosfotransferasas (Aceptor de Grupo Alcohol)/antagonistas & inhibidores , Fosfotransferasas (Aceptor de Grupo Alcohol)/química , Fosfotransferasas (Aceptor del Grupo Fosfato) , Polifosfatos/metabolismo , Relación Estructura-ActividadRESUMEN
Toxin-antitoxin (TA) complexes play an important role in stress responses and programmed cell death in bacteria. The RelB-RelE toxin antitoxin system is well studied in Escherichia coli. In this study, we used combined in silico and in vitro approaches to study a novel Xn-RelT toxin from Xenorhabdus nematophila bearing its own antitoxin Xn-RelAT-a RelB homolog of E. coli. The structure for this toxin-antitoxin pair is yet unknown. We generated homology-based models of X. nematophila RelT toxin and antitoxin. The deduced models were further characterized for protein-nucleic acid, protein-protein interactions and gene ontology. A detrimental effect of recombinant Xn-RelT on host E. coli was determined through endogenous toxicity assay. When expressed from a isopropyl ß-D-1-thiogalactopyranoside-regulated LacZ promoter, Xn-RelT toxin showed a toxic effect on E. coli cells. These observations imply that the conditional cooperativity governing the Xn-RelT TA operon in X. nematophila plays an important role in stress management and programmed cell death.
RESUMEN
Bacterial toxin-antitoxin (TA) complexes induce programmed cell death and also function to relieve cell from stress by various response mechanisms. Escherichia coli RelB-RelE TA complex consists of a RelE toxin functionally counteracted by RelB antitoxin. In the present study, a novel homolog of RelE toxin designated as Xn-relE toxin from Xenorhabdus nematophila possessing its own antitoxin designated as Xn-relEAT has been identified. Expression and purification of recombinant proteins under native conditions with GST and Ni-NTA chromatography prove the existence of novel TA module. The expression of recombinant Xn-relE under tightly regulated ara promoter in E. coli Top 10 cells confirms its toxic nature in endogenous toxicity assay. The neutralization activity in endogenous toxicity assay by Xn-relEAT antitoxin confirms its antidote nature when studying the whole TA operon under ara regulated promoter. This study promotes newly discovered TA module to be regarded as important as other proteins of type II toxin-antitoxin system.