ABSTRACT
ß-Lactamase inhibitors (clavulanic acid, sulbactam, and tazobactam) contribute significantly to the longevity of the ß-lactam antibiotics used to treat serious infections. In the quest to design more potent compounds and to understand the mechanism of action of known inhibitors, 6ß-(hydroxymethyl)penicillanic acid sulfone (6ß-HM-sulfone) was tested against isolates expressing the class A TEM-1 ß-lactamase and a clinically important variant of the AmpC cephalosporinase of Pseudomonas aeruginosa, PDC-3. The addition of the 6ß-HM-sulfone inhibitor to ampicillin was highly effective. 6ß-HM-sulfone inhibited TEM-1 with an IC(50) of 12 ± 2 nM and PDC-3 with an IC(50) of 180 ± 36 nM, and displayed lower partition ratios than commercial inhibitors, with partition ratios (k(cat)/k(inact)) equal to 174 for TEM-1 and 4 for PDC-3. Measured for 20 h, 6ß-HM-sulfone demonstrated rapid, first-order inactivation kinetics with the extent of inactivation being related to the concentration of inhibitor for both TEM-1 and PDC-3. Using mass spectrometry to gain insight into the intermediates of inactivation of this inhibitor, 6ß-HM-sulfone was found to form a major adduct of +247 ± 5 Da with TEM-1 and +245 ± 5 Da with PDC-3, suggesting that the covalently bound, hydrolytically stabilized acyl-enzyme has lost a molecule of water (HOH). Minor adducts of +88 ± 5 Da with TEM-1 and +85 ± 5 Da with PDC-3 revealed that fragmentation of the covalent adduct can result but appeared to occur slowly with both enzymes. 6ß-HM-sulfone is an effective and versatile ß-lactamase inhibitor of representative class A and C enzymes.
Subject(s)
Anti-Bacterial Agents/pharmacology , Bacterial Proteins/metabolism , Sulbactam/analogs & derivatives , Sulbactam/pharmacology , beta-Lactamases/metabolism , Anti-Bacterial Agents/chemistry , Bacterial Proteins/antagonists & inhibitors , Bacterial Proteins/genetics , Catalytic Domain , Computer Simulation , Escherichia coli/drug effects , Escherichia coli/enzymology , Escherichia coli/genetics , Models, Molecular , Molecular Structure , Protein Conformation , Pseudomonas aeruginosa/enzymology , Sulbactam/chemistry , beta-Lactamase Inhibitors , beta-Lactamases/geneticsABSTRACT
Previous studies indicate that STAT5 expression is required for mast cell development, survival, and IgE-mediated function. STAT5 tyrosine phosphorylation is swiftly and transiently induced by activation of the high affinity IgE receptor, FcεRI. However, the mechanism for this mode of activation remains unknown. In this study we observed that STAT5 co-localizes with FcεRI in antigen-stimulated mast cells. This localization was supported by cholesterol depletion of membranes, which ablated STAT5 tyrosine phosphorylation. Through the use of various pharmacological inhibitors and murine knock-out models, we found that IgE-mediated STAT5 activation is dependent upon Fyn kinase, independent of Syk, PI3K, Akt, Bruton's tyrosine kinase, and JAK2, and enhanced in the context of Lyn kinase deficiency. STAT5 immunoprecipitation revealed that unphosphorylated protein preassociates with Fyn and that this association diminishes significantly during mast cell activation. SHP-1 tyrosine phosphatase deficiency modestly enhanced STAT5 phosphorylation. This effect was more apparent in the absence of Gab2, a scaffolding protein that docks with multiple negative regulators, including SHP-1, SHP-2, and Lyn. Targeting of STAT5A or B with specific siRNA pools revealed that IgE-mediated mast cell cytokine production is selectively dependent upon the STAT5B isoform. Altogether, these data implicate Fyn as the major positive mediator of STAT5 after FcεRI engagement and demonstrate importantly distinct roles for STAT5A and STAT5B in mast cell function.
Subject(s)
Cytokines/biosynthesis , Mast Cells/metabolism , Receptors, IgE/metabolism , STAT5 Transcription Factor/metabolism , Signal Transduction/physiology , Adaptor Proteins, Signal Transducing , Agammaglobulinaemia Tyrosine Kinase , Animals , Cells, Cultured , Cholesterol/genetics , Cholesterol/metabolism , Cytokines/genetics , Janus Kinase 2/genetics , Janus Kinase 2/metabolism , Mast Cells/cytology , Mice , Mice, Knockout , Phosphatidylinositol 3-Kinases/genetics , Phosphatidylinositol 3-Kinases/metabolism , Phosphoproteins/genetics , Phosphoproteins/metabolism , Phosphorylation/physiology , Protein Tyrosine Phosphatase, Non-Receptor Type 6/genetics , Protein Tyrosine Phosphatase, Non-Receptor Type 6/metabolism , Protein-Tyrosine Kinases/genetics , Protein-Tyrosine Kinases/metabolism , Proto-Oncogene Proteins c-akt/genetics , Proto-Oncogene Proteins c-akt/metabolism , Proto-Oncogene Proteins c-fyn/genetics , Proto-Oncogene Proteins c-fyn/metabolism , Receptors, IgE/genetics , STAT5 Transcription Factor/genetics , src-Family Kinases/genetics , src-Family Kinases/metabolismABSTRACT
In order to evaluate the importance of a hydrogen-bond donating substituent in the design of ß-lactamase inhibitors, a series of C6-substituted penicillin sulfones, lacking a C2' substituent, and having an sp(3) hybridized C6, was prepared and evaluated against a representative classes A and C ß-lactamases. It was found that a C6 hydrogen-bond donor is necessary for good inhibitory activity, but that this feature alone is not sufficient in this series of C6ß-substituted penicillin sulfones. Other factors which may impact the potency of the inhibitor include the steric bulk of the C6 substituent (e.g., methicillin sulfone) which may hinder recognition in the class A ß-lactamases, and also high similarity to the natural substrates (e.g., penicillin G sulfone) which may render the prospective inhibitor a good substrate of both classes of enzyme. The best inhibitors had non-directional hydrogen-bonding substituents, such as hydroxymethyl, which may allow sufficient conformational flexibility of the acyl-enzyme for abstraction of the C6 proton by E166 (class A), thus promoting isomerization to the ß-aminoacrylate as a stabilized acyl-enzyme.
