13 research outputs found

    A Dynamic Pathway for Calcium-Independent Activation of CaMKII by Methionine Oxidation

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    SummaryCalcium/calmodulin (Ca2+/CaM)-dependent protein kinase II (CaMKII) couples increases in cellular Ca2+ to fundamental responses in excitable cells. CaMKII was identified over 20 years ago by activation dependence on Ca2+/CaM, but recent evidence shows that CaMKII activity is also enhanced by pro-oxidant conditions. Here we show that oxidation of paired regulatory domain methionine residues sustains CaMKII activity in the absence of Ca2+/CaM. CaMKII is activated by angiotensin II (AngII)-induced oxidation, leading to apoptosis in cardiomyocytes both in vitro and in vivo. CaMKII oxidation is reversed by methionine sulfoxide reductase A (MsrA), and MsrA−/− mice show exaggerated CaMKII oxidation and myocardial apoptosis, impaired cardiac function, and increased mortality after myocardial infarction. Our data demonstrate a dynamic mechanism for CaMKII activation by oxidation and highlight the critical importance of oxidation-dependent CaMKII activation to AngII and ischemic myocardial apoptosis

    Compound Efflux in Helicobacter pylori

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    Susceptibility testing with a variety of structurally unrelated compounds showed that hefC in Helicobacter pylori is involved in multidrug efflux. This efflux was shown to depend on the proton motive force, as demonstrated by ethidium bromide accumulation experiments. Thus, H. pylori contains an active multidrug efflux mechanism

    Discovery of Novel DNA Gyrase Inhibiting Spiropyrimidinetriones: Benzisoxazole Fusion with N‑Linked Oxazolidinone Substituents Leading to a Clinical Candidate (ETX0914)

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    A novel class of bacterial type-II topoisomerase inhibitor displaying a spiropyrimidinetrione architecture fused to a benzisoxazole scaffold shows potent activity against Gram-positive and fastidious Gram-negative bacteria. Here, we describe a series of <i>N</i>-linked oxazolidinone substituents on the benzisoxazole that improve upon the antibacterial activity of initially described compounds of the class, show favorable PK properties, and demonstrate efficacy in an in vivo Staphylococcus aureus infection model. Inhibition of the topoisomerases DNA gyrase and topoisomerase IV from both Gram-positive and a Gram-negative organisms was demonstrated. Compounds showed a clean in vitro toxicity profile, including no genotoxicity and no bone marrow toxicity at the highest evaluated concentrations or other issues that have been problematic for some fluoroquinolones. Compound <b>1u</b> was identified for advancement into human clinical trials for treatment of uncomplicated gonorrhea based on a variety of beneficial attributes including the potent activity and the favorable safety profile

    Novel DNA Gyrase Inhibiting Spiropyrimidinetriones with a Benzisoxazole Scaffold: SAR and in Vivo Characterization

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    The compounds described herein with a spirocyclic architecture fused to a benzisoxazole ring represent a new class of antibacterial agents that operate by inhibition of DNA gyrase as corroborated in an enzyme assay and by the inhibition of precursor thymidine into DNA during cell growth. Activity resided in the configurationally lowest energy (2<i>S</i>,4<i>R</i>,4a<i>R</i>) diastereomer. Highly active compounds against Staphylococcus aureus had sufficiently high solubility, high plasma protein free fraction, and favorable pharmacokinetics to suggest that in vivo efficacy could be demonstrated, which was realized with compound (−)-<b>1</b> in S. aureus mouse infection models. A high drug exposure NOEL on oral dosing in the rat suggested that a high therapeutic margin could be achieved. Importantly, (−)-<b>1</b> was not cross-resistant with other DNA gyrase inhibitors such as fluoroquinolone and aminocoumarin antibacterials. Hence, this class shows considerable promise for the treatment of infections caused by multidrug resistant bacteria, including S. aureus

    <i>In Vivo</i> Validation of Thymidylate Kinase (TMK) with a Rationally Designed, Selective Antibacterial Compound

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    There is an urgent need for new antibacterials that pinpoint novel targets and thereby avoid existing resistance mechanisms. We have created novel synthetic antibacterials through structure-based drug design that specifically target bacterial thymidylate kinase (TMK), a nucleotide kinase essential in the DNA synthesis pathway. A high-resolution structure shows compound TK-666 binding partly in the thymidine monophosphate substrate site, but also forming new induced-fit interactions that give picomolar affinity. TK-666 has potent, broad-spectrum Gram-positive microbiological activity (including activity against methicillin-resistant <i>Staphylococcus aureus</i> and vancomycin-resistant <i>Enterococcus</i>), bactericidal action with rapid killing kinetics, excellent target selectivity over the human ortholog, and low resistance rates. We demonstrate <i>in vivo</i> efficacy against <i>S. aureus</i> in a murine infected-thigh model. This work presents the first validation of TMK as a compelling antibacterial target and provides a rationale for pursuing novel clinical candidates for treating Gram-positive infections through TMK