Unraveling topoisomerase IA gate dynamics in presence of PPEF and its preclinical evaluation against multidrug-resistant pathogens.

Type IA topoisomerases maintain DNA topology by cleaving ssDNA and relaxing negative supercoils. The inhibition of its activity in bacteria prevents the relaxation of negative supercoils, which in turn impedes DNA metabolic processes leading to cell death. Using this hypothesis, two bisbenzimidazoles, PPEF and BPVF are synthesized, selectively inhibiting bacterial TopoIA and TopoIII. PPEF stabilizes the topoisomerase and topoisomerase-ssDNA complex, acts as an interfacial inhibitor. PPEF display high efficacy against ~455 multi-drug resistant gram positive and negative bacteria. To understand molecular mechanism of inhibition of TopoIA and PPEF, accelerated MD simulation is carried out, and results suggested that PPEF binds, stabilizes the closed conformation of TopoIA with -6Kcal/mol binding energy and destabilizes the binding of ssDNA. The TopoIA gate dynamics model can be used as a tool to screen TopoIA inhibitors as therapeutic candidates. PPEF and BPVF cause cellular filamentation and DNA fragmentation leading to bacterial cell death. PPEF and BPVF show potent efficacy against systemic and neutropenic mouse models harboring E. coli, VRSA, and MRSA infection without cellular toxicity.

Synthesis and Biological Evaluation of Novel 1H-Benzo[d]imidazole Derivatives as Potential Anticancer Agents Targeting Human Topoisomerase I.

Small molecules that modulate biological functions are targets of modern-day drug discovery efforts. A new series of novel 1H-benzo[d]imidazoles (BBZs) were designed and synthesized with different functional groups at the phenyl ring and variable lengths of the alkyl chain at the piperazine end as anticancer agents. We identified human topoisomerase I (Hu Topo I) as a probable target of these molecules through a computational study and DNA relaxation assay, a functional assay of the Hu Topo I enzyme. UV absorption, fluorescence, and circular dichroism spectroscopy were used to study interactions between BBZ and DNA. Out of 16 compounds, 11a, 12a, and 12b showed strong binding affinity and thermal stabilization of AT sequence-specific DNA. BBZs were screened against a panel of 60 human cancer cell lines at National Cancer Institute, USA. Most potent molecules 11a, 12a, and 12b showed 50% growth inhibition (GI50) in a concentration range from 0.16 to 3.6 µM cancer cells. Moreover, 12b showed 50% inhibition of the relaxation of DNA by Hu Topo I at 16 µM. Furthermore, flow cytometry revealed that 11a, 12a, and 12b cause prominent G2M arrest of cancer cells. In view of the above, we propose that 12b deserves to be further evaluated for its therapeutic use as an anticancer agent.

PPEF: A bisbenzimdazole potent antimicrobial agent interacts at acidic triad of catalytic domain of E. coli topoisomerase IA.

BACKGROUND: Topoisomerase is a well known target to develop effective antibacterial agents. In pursuance of searching novel antibacterial agents, we have established a novel bisbenzimidazole (PPEF) as potent E. coli topoisomerase IA poison inhibitor. METHODS: In order to gain insights into the mechanism of action of PPEF and understanding protein-ligand interactions, we have produced wild type EcTopo 67 N-terminal domain (catalytic domain) and its six mutant proteins at acidic triad (D111, D113, E115). The DDE motif is replaced by alanine (A) to create three single mutants: D111A, D113A, E115A and three double mutants: D111A-D113A, D113A-E115A and D111A-E115A. RESULTS: Calorimetric study of PPEF with single mutants showed 10 fold lower affinity than that of wild type EcTopo 67 (7.32 × 106 M-1for wild type, 0.89 × 106 M-1for D111A) and 100 fold lower binding with double mutant D113A-E115A (0.02 × 106 M-1) was observed. The mutated proteins showed different CD signature as compared to wild type protein. CD and fluorescence titrations were done to study the interaction between EcTopo 67 and ligands. Molecular docking study validated that PPEF has decreased binding affinity towards mutated enzymes as compared to wild type. CONCLUSION: The overall study reveals that PPEF binds to D113 and E115 of acidic triad of EcTopo 67. Point mutations decrease binding affinity of PPEF towards DDE motif of topoisomerase. GENERAL SIGNIFICANCE: This study concludes PPEF as poison inhibitor of E. coli Topoisomerase IA, which binds to acidic triad of topoisomerase IA, responsible for its function. PPEF can be considered as therapeutic agent against bacteria.

Synergistic efficacy of Bisbenzimidazole and Carbonyl Cyanide 3-Chlorophenylhydrazone combination against MDR bacterial strains.

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.

Topoisomerases: Resistance versus Sensitivity, How Far We Can Go?

DNA topoisomerases are ubiquitously present remarkable molecular machines that help in altering topology of DNA in living cells. The crucial role played by these nucleases during DNA replication, transcription, and recombination vis-à-vis less sequence similarity among different species makes topoisomerases unique and attractive targets for different anticancer and antibacterial drugs. However, druggability of topoisomerases by the existing class of molecules is increasingly becoming questationable due to resistance development predominated by mutations in the corresponding genes. The current scenario facing a decline in the development of new molecules further comprises an important factor that may challenge topoisomerase-targeting therapy. Thus, it is imperative to wisely use the existing inhibitors lest with this rapid rate of losing grip over the target we may not go too far. Furthermore, it is important not only to design new molecules but also to develop new approaches that may avoid obstacles in therapies due to multiple resistance mechanisms. This review provides a succinct account of different classes of topoisomerase inhibitors, focuses on resistance acquired by mutations in topoisomerases, and discusses the various approaches to increase the efficacy of topoisomerase inhibitors. In a later section, we also suggest the possibility of using bisbenzimidazoles along with efflux pump inhibitors for synergistic bactericidal effects.

Hematopoietic Effect of Amaranthus cruentus Extract on Phenylhydrazine-Induced Toxicity in Rats.

Amaranthus cruentus (Amaranthaceae) is one of the popularly grown leafy vegetables in the Indian subcontinent. Leaves of the plant are rich in polyphenols, tannins, flavonoids, steroids, terpenoids, saponins, and betalains. The plant also contains rich amounts of protein, calcium, iron, vitamins A, E, and C, and folic acid. The present work was undertaken to evaluate the antianemic effect of Amaranthus cruentus. Ethanol extract of Amaranthus cruentus was prepared. Acute oral toxicity of the extract was determined by Organization for Economic Cooperation and Development (OECD) Guideline 423. Doses of 200 and 400 mg/kg were used in the present study. Phenylhydrazine (60 mg/kg, intraperitoneal injection for three consecutive days) was used to induce anemia in rats. After anemia induction, animals were treated with standard preparation and extract. Amaranthus cruentus extract significantly aided in restoring the levels of red blood cells, white blood cells (WBCs), and hemoglobin. There was also an increase in hematocrit. Thus, it can be concluded that Amaranthus cruentus is a rich source of phytochemicals that are responsible for demonstrating hematopoietic effects. Isolation and structure elucidation of constituents, responsible for antianemic activity, is necessary to affirm the aforementioned effect.