Novel antibacterial drugs that are effective against infections caused by multidrug resistant pathogens are urgently needed. In a previous report, we have shown that tetrahydropyran-based inhibitors of bacterial type II topoisomerases (DNA gyrase and topoisomerase IV) display potent antibacterial activity and exhibit no target-mediated cross-resistance with fluoroquinolones. During the course of our optimization program, lead compound 5 was deprioritized due to adverse findings in cardiovascular safety studies. In the effort of mitigating these findings and optimizing further the pharmacological profile of this class of compounds, we have identified a subseries of tetrahydropyran-based molecules that are potent DNA gyrase and topoisomerase IV inhibitors and display excellent antibacterial activity against Gram positive pathogens, including clinically relevant resistant isolates. One representative of this class, compound 32d, elicited only weak inhibition of hERG K(+) channels and hNaV1.5 Na(+) channels, and no effects were observed on cardiovascular parameters in anesthetized guinea pigs. In vivo efficacy in animal infection models has been demonstrated against Staphylococcus aureus and Streptococcus pneumoniae strains.
The proteinVirB8 plays a critical role in the assembly and function of the Agrobacterium tumefaciens virB type IV secretion system (T4SS). The structure of the periplasmic domain of both A. tumefaciens and Brucella suis VirB8 has been determined, and site-directed mutagenesis has revealed amino acids involved in the dimerization of VirB8 and interactions with VirB4 and VirB10. We have shown previously that TraJ, the VirB8 homologue from pSB102, and the chimeric protein TraJB8, encompassing the cytoplasmic and transmembrane (TM) domains of TraJ and the periplasmic domain of VirB8, were unable to complement a B. suis mutant containing an in-frame deletion of the virB8 gene. This suggested that the presence of the TraJ cytoplasmic and TM domains could block VirB8 dimerization or assembly in the inner membrane. By bacterial two-hybrid analysis, we found that VirB8, TraJ, and the chimeras can all interact to form both homo-and heterodimers. However, the presence of the TM domain of TraJ resulted in much stronger interactions in both the homo-and heterodimers. We expressed the wild-type and chimeric proteins in wild-type B. suis. The presence of proteins carrying the TM domain of TraJ had a dominant negative effect, leading to complete loss of virulence. This suggests that the T4SS is a dynamic structure and that strong interactions block the spatial flexibility required for correct assembly and function.
Under pathological conditions, the purinergic P2X7 receptor is activated by elevated concentrations of extracellular ATP. Thereby, the receptor forms a slowly dilating pore, allowing cations and, upon prolonged stimulation, large molecules to enter the cell. This process has a strong impact on cell signaling, metabolism, and viability. This study aimed to establish a link between gradual P2X7 activation and pharmacological endpoints including oxidative stress, hydrogen peroxide generation, and cytotoxicity. Mechanisms of cellular stress and cytotoxicity were studied in P2X7-transfected HEK293 cells. We performed real-time monitoring of metabolic and respiratory activity of cells expressing the P2X7-receptor protein using a cytosensor system. Agonistic effects were monitored using exogenously applied ATP or the stable analogue BzATP. Oxidative stress induced by ATP or BzATP in target cells was monitored by hydrogen peroxide release in human mononuclear blood cells. P2X7-receptor activation was studied by patch-clamp experiments using a primary mouse microglia cell line. Stimulation of the P2X7 receptor leads to ion influx, metabolic activation of target cells, and ultimately cytotoxicity. Conversion of the P2X7 receptor from a small cation channel to a large pore occurring under prolonged stimulation can be monitored in real time covering a time frame of milliseconds to hours. Selectivity of the effects can be demonstrated using the selective P2X7-receptor antagonist AZD9056. Our findings established a direct link between P2X7-receptor activation by extracellular ATP or BzATP and cellular events culminating in cytotoxicity. Mechanisms of toxicity include metabolic and oxidative stress, increase in intracellular calcium concentration and disturbance of mitochondrial membrane potential. Mitochondrial toxicity is suggested to be a key event leading to cell death.
Introduction: Cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CM) form spontaneously beating syncytia in-vitro. We evaluated whether hiPSC-CM are a compelling model of human cardiac pharmacology useful for early drug development. Methods: We measured hiPSC-CM beating frequency using Ca-sensitive dyes and a high-throughput screening system. We quantified the effects of 640 drugs with various structures and pharmacologies. Results: When tested at 1 µM, most drugs without direct effects on heart rhythm or with effects at high concentrations do not change frequency, indicating specificity. In contrast, the preparation detects compounds with direct activity on heart rhythm, demonstrating sensitivity. In particular, β-adrenergic agonists increase frequency and the model differentiates β2 from β1 agonists, as well as partial from full agonists. Phosphodiesterase inhibitors have subtype-specific actions and PDE4 is particularly important in controlling frequency. The preparation is sensitive to cardiac ion channel blockers: L-type calcium channel blockers, Class-I and Class-III antiarrhythmics change frequency but drugs acting on K ATP channels do not. The assay detects compounds blocking the cardiac rapid delayed-rectifier K channel and is an alternative to the classic "hERG test".Conclusion: hiPSC-CM are a useful in-vitro cardiac model in drug development since they respond appropriately to drugs that modify heart rate in humans.
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