The identification and characterization of novel antibacterial compounds via target-based and whole cell screening approaches

The emergence of multidrug-resistant bacterial infections in both the clinical setting and the community has created an environment in which the development of novel antibacterial compounds is necessary to keep dangerous infections at bay. While the derivatization of existing antibiotics by pharmaceutical companies has so far been successful at achieving this end, this strategy is short-term, and the discovery of antibacterials with novel scaffolds would be a greater contribution to the fight of multidrug-resistant infections. Described herein is the application of both target-based and whole cell screening strategies to identify novel antibacterial compounds. In a target-based approach, we sought small-molecule disruptors of the MazEF toxin-antitoxin protein complex. A lack of facile, continuous assays for this target required the development of a fluorometric assay for MazF ribonuclease activity. This assay was employed to further characterize the activity of the MazF enzyme and was used in a screening effort to identify disruptors of the MazEF complex. In addition, by employing a whole cell screening approach, we identified two compounds with potent antibacterial activity. Efforts to characterize the in vitro antibacterial activities displayed by these compounds and to identify their modes of action are described.

Molecular mechanisms involved in cell response to mechanical forces

New devices were designed to generate a localized mechanical vibration of flexible gels where human umbilical vein endothelial cells (HUVECs) were cultured. The stimulation setups were able to apply relatively large strains (30%~50%) at high temporal frequencies (140~207 Hz) in a localized subcellular region. One of the advantages of this technique was to be less invasive to the innate cellular functions because there was no direct contact between the stimulating probe and the cell body. A mechanical vibration induced by the device in the substrate gel where cells were seeded could mainly cause global calcium responses of the cells. This global response was initiated by the influx of calcium across the stretch-activated channels in the plasma membrane. The subsequent production of inositol triphosphate (IP3) via phospholipase C (PLC) activation triggered the calcium release from the endoplasmic reticulum (ER) to cause a global intracellular calcium fluctuation over the whole cell body. This global calcium response was also shown to depend on actomyosin contractility and F-actin integrity, probably controlling the membrane stretch-activated channels. The localized nature of the stimulation is one of the most important features of these new designs as it allowed the observation of the calcium signaling propagation by ER calcium release. The next step was to focus on the calcium influx, more specifically the TRPM7 channels. As TRPM7 expression may modulate cell adhesion, an adhesion assay was developed and tested on HUVECs seeded on gel substrates with different treatments: normal treatment on gels showed highest attachment rate, followed by the partially treated gels (only 5% of usual fibronectin amount) and untreated gels, with the lowest attachment rate. The trend of the attachment rates correlated to the magnitude of the calcium signaling observed after mechanical stimulation. TRPM7 expression inhibition by siRNA caused an increased attachment rate when compared to both control and non-targeting siRNA-treated cells, but resulted in an actual weaker response in terms of calcium signaling. It suggests that TRPM7 channels are indeed important for the calcium signaling in response to mechanical stimulation. A complementary study was also conducted consisting in the mechanical stimulation of a dissected Drosophila embryo. Although ionomycin treatment showed calcium influx in the tissue, the mechanical stimulation delivered as a vertical vibration did not elicited calcium signaling in response. One possible reason is the dissection procedure causing desensitization of the tissue due to the scrapings and manipulations to open the embryo.