Bacterial DNA gyrase is a well-known and validated target in the design of antibacterial drugs. However, inhibitors of its ATP binding subunit, DNA gyrase B (GyrB), have so far not reached clinical use. In the present study, three different series of N-phenyl-4,5-dibromopyrrolamides and N-phenylindolamides were designed and prepared as potential DNA gyrase B inhibitors. The IC50 values of compounds on DNA gyrase from Escherichia coli were in the low micromolar range, with the best compound, (4-(4,5-dibromo-1H-pyrrole-2-carboxamido)benzoyl)glycine (18a), displaying an IC50 of 450 nM. For this compound, a high-resolution crystal structure in complex with E. coli DNA gyrase B was obtained, revealing details of its binding mode within the active site. The binding affinities of three compounds with GyrB were additionally evaluated by surface plasmon resonance, and the results were in good agreement with the determined enzymatic activities. For the most promising compounds, the inhibitory activities against DNA gyrase from Staphylococcus aureus and topoisomerases IV from E. coli and S. aureus were determined. Antibacterial activities of the most potent compounds of each series were evaluated against two Gram-positive and two Gram-negative bacterial strains. The results obtained in this study provide valuable information on the binding mode and structure-activity relationship of N-phenyl-4,5-dibromopyrrolamides and N-phenylindolamides as promising classes of ATP competitive GyrB inhibitors.
The human inducible phospho-fructokinase bisphosphatase isoform 3, PFKFB3, is a crucial regulatory node in the cellular metabolism. The enzyme is an important modulator regulating the intracellular fructose-2,6-bisphosphate level. PFKFB3 is a bifunctional enzyme with an exceptionally high kinase to phosphatase ratio around 740:1. Its kinase activity can be directly inhibited by small molecules acting directly on the kinase active site. On the other hand, here we propose an innovative and indirect strategy for the modulation of PFKFB3 activity, achieved through allosteric bisphosphatase activation. A library of small peptides targeting an allosteric site was discovered and synthesized. The binding affinity was evaluated by microscale thermophoresis (MST). Furthermore, a LC-MS/MS analytical method for assessing the bisphosphatase activity of PFKFB3 was developed. The new method was applied for measuring the activation on bisphosphatase activity with the PFKFB3-binding peptides. The molecular mechanical connection between the newly discovered allosteric site to the bisphosphatase activity was also investigated using both experimental and computational methods.
Depsipeptides are biologically active peptide derivatives that possess a high therapeutic interest. The development of depsipeptide mimics characterized by a chemical diversity could lead to compounds with enhanced features and activity. In this work, an on resin multicomponent procedure for the synthesis of amidino depsipeptide mimics is described. This approach exploits a metal-free 1,3-dipolar cycloaddition of cyclopentanone -proline enamines and sulfonylazides. In this reaction, the obtained primary cycloadduct undergoes a ring opening and molecular rearrangement giving access to a linear sulfonyl amidine functionalized with both a peptide chain and a diazoalkane. The so obtained diazo function "one pot" reacts with the carboxylic group of N-Fmoc protected amino acids leading to amidino depsipeptide mimics possessing a C4 aliphatic chain.An important advantage of this procedure is the possibility to easily obtain amidino functionalized derivatives that are proteolitically stable peptide bond bioisosters. Moreover, the conformational freedom given by the alkyl chain could promote the obtainment of cyclic depsipeptide with a stabilized secondary structure as demonstrated with both in silico calculations and experimental conformational studies. Finally, labelled depsipeptide mimics can be also synthesized using a fluorescent sulfonylazide in the multicomponent reaction.
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