ABSTRACT
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.
Subject(s)
Acinetobacter baumannii/enzymology , Acinetobacter baumannii/metabolism , Anti-Bacterial Agents/pharmacology , Phosphotransferases (Phosphate Group Acceptor)/drug effects , Phosphotransferases (Phosphate Group Acceptor)/metabolism , Virulence Factors/genetics , Acid Anhydride Hydrolases/metabolism , Acinetobacter Infections/microbiology , Acinetobacter baumannii/drug effects , Acinetobacter baumannii/genetics , Acyl-Butyrolactones/metabolism , Biofilms/growth & development , Cloning, Molecular , Drug Resistance, Multiple, Bacterial/drug effects , Gene Expression Regulation, Bacterial , Microbial Sensitivity Tests , Molecular Docking Simulation , Phosphotransferases (Phosphate Group Acceptor)/chemistry , Phosphotransferases (Phosphate Group Acceptor)/genetics , Polyphosphates/metabolism , Sequence Analysis , Virulence/geneticsABSTRACT
By targeting the thiamin diphosphate (ThDP) binding site of Escherichia coli (E. coli) pyruvate dehydrogenase multienzyme complex E1 (PDHc E1), a series of novel 'open-chain' classes of ThDP analogs A, B, and C with N-acylhydrazone moieties was designed and synthesized to explore their activities against E. coli PHDc E1 in vitro and their inhibitory activity against microbial diseases were further evaluated in vivo. As a result, A1-23 exhibited moderate to potent inhibitory activities against E. coli PDHc E1 (IC50=0.15-23.55µM). The potent inhibitors A13, A14, A15, C2, had strong inhibitory activities with IC50 values of 0.60, 0.15, 0.39 and 0.34µM against E. coli PDHc E1 and with good enzyme-selective inhibition between microorganisms and mammals. Especially, the most powerful inhibitor A14 could 99.37% control Xanthimonas oryzae pv. Oryzae. Furthermore, the binding features of compound A14 within E. coli PDHc E1 were investigated to provide useful insights for the further construction of new inhibitor by molecular docking, site-directed mutagenesis, and enzymatic assays. The results indicated that A14 had most powerful inhibition against E. coli PDHc E1 due to the establishment of stronger interaction with Glu571, Met194, Glu522, Leu264 and Phe602 at active site of E.coli PDHc E1. It could be used as a lead compound for further optimization, and may have potential as a new microbicide.
Subject(s)
Drug Delivery Systems , Escherichia coli/drug effects , Molecular Docking Simulation , Phosphotransferases (Phosphate Group Acceptor)/chemistry , Pyrimidines/chemistry , Pyrimidines/pharmacology , Pyruvate Dehydrogenase Complex/antagonists & inhibitors , Animals , Anti-Infective Agents/chemistry , Anti-Infective Agents/pharmacology , Binding Sites , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Escherichia coli/enzymology , Inhibitory Concentration 50 , Phosphotransferases (Phosphate Group Acceptor)/drug effects , Structure-Activity Relationship , SwineABSTRACT
Campylobacter jejuni is the most prevalent cause of bacterial gastroenteritis worldwide. Polyphosphate kinases 1 and 2 (PPK1 and PPK2) regulate several cellular processes, including the biosynthesis of the bacterial cell wall. Despite their importance, whether PPK1 and PPK2 modulate the composition of C. jejuni outer membrane constituents (OMCs) and consequently impact its interaction with host cells remains unknown. Our comparative analysis between C. jejuni wild type, Δppk1, and Δppk2 strains showed qualitative and quantitative differences in the total OMC composition among these strains. Importantly, these OMC variations observed on the C. jejuni polyphosphate kinase mutants are directly related to their capacity to invade, survive, and alter the immune response of intestinal epithelial cells in vitro. Specifically, sub-fractionation of the C. jejuni OMC indicated that OMC proteins are uniquely associated with bacterial invasion, whereas C. jejuni OMC proteins, lipids, and lipoglycans are all associated with C. jejuni intracellular survival. This study provides new insights regarding the function of polyphosphate kinases and their role in C. jejuni infection.
Subject(s)
Anti-Infective Agents/pharmacology , Campylobacter Infections/microbiology , Campylobacter jejuni/cytology , Campylobacter jejuni/pathogenicity , Epithelial Cells/microbiology , Gastroenteritis/microbiology , Phosphotransferases (Phosphate Group Acceptor)/metabolism , Campylobacter Infections/drug therapy , Campylobacter jejuni/drug effects , Cell Line , Epithelial Cells/drug effects , Gastroenteritis/drug therapy , Humans , In Vitro Techniques , Interleukin-8/metabolism , Molecular Targeted Therapy/trends , Phosphotransferases (Phosphate Group Acceptor)/drug effectsABSTRACT
PURPOSE: To evaluate the effects and mechanism of aminoimidazole carboxamide ribonucleotide (AICAR), an AMP-dependent kinase (AMPK) activator, on the growth of uveal melanoma cell lines. METHODS: Four different cell lines were treated with AICAR (1-4 mM). Cell growth was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay. Cell cycle analysis was conducted by flow cytometry; additionally, expression of cell-cycle control proteins, cell growth transcription factors, and downstream effectors of AMPK were determined by RT-PCR and Western blot. RESULTS: Aminoimidazole carboxamide ribonucleotide inhibited cell growth, induced S-phase arrest, and led to AMPK activation. Aminoimidazole carboxamide ribonucleotide treatment was associated with inhibition of eukaryotic translation initiation factor 4E-BP1 phosphorylation, a marker of mammalian target of rapamycin (mTOR) pathway activity. Aminoimidazole carboxamide ribonucleotide treatment was also associated with downregulation of cyclins A and D, but had minimal effects on the phosphorylation of ribosomal protein S6 or levels of the macroautophagy marker LC3B. The effects of AICAR were abolished by treatment with dipyridamole, an adenosine transporter inhibitor that blocks the entry of AICAR into cells. Treatment with adenosine kinase inhibitor 5-iodotubericidin, which inhibits the conversion of AICAR to its 5'-phosphorylated ribotide 5-aminoimidazole-4-carboxamide-1-D-ribofuranosyl-5'-monophosphate (ZMP; the direct activator of AMPK), reversed most of the growth-inhibitory effects, indicating that some of AICAR's antiproliferative effects are mediated at least partially through AMPK activation. CONCLUSIONS: Aminoimidazole carboxamide ribonucleotide inhibited uveal melanoma cell proliferation partially through activation of the AMPK pathway and downregulation of cyclins A1 and D1.
Subject(s)
Aminoimidazole Carboxamide/analogs & derivatives , Enzyme Activation/drug effects , Gene Expression Regulation, Neoplastic , Melanoma/genetics , Phosphotransferases (Phosphate Group Acceptor)/metabolism , RNA, Neoplasm/genetics , Ribonucleotides/pharmacology , TOR Serine-Threonine Kinases/genetics , Uveal Neoplasms/genetics , Aminoimidazole Carboxamide/pharmacology , Blotting, Western , Cell Cycle/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Flow Cytometry , Humans , Melanoma/drug therapy , Melanoma/enzymology , Phosphotransferases (Phosphate Group Acceptor)/drug effects , Real-Time Polymerase Chain Reaction , TOR Serine-Threonine Kinases/metabolism , Uveal Neoplasms/drug therapy , Uveal Neoplasms/enzymologyABSTRACT
Ring-B derivatization of totarol (1) afforded the series of compounds 2-22 which were screened in vitro against: beta-lactamase-positive and high level gentamycin-resistant Enterococcus faecalis, penicillin-resistant Streptococcus pneumoniae, methicillin-resistant Staphylococcus aureus (MRSA), and multiresistant Klebsiella pneumoniae. Several of the derivatives retained much of the antibacterial activity of totatol against the first three of these organisms (all gram-positive), but none was more active. The gram-negative Klebsiella was resistant to all compounds examined. Totarol (1) was shown to uncouple oxidative phosphorylation in isolated mitochondria at 50 microM.