Structure-based discovery and in vitro validation of inhibitors of chloride intracellular channel 4 protein.

The use of computer-aided methods have continued to propel accelerated drug discovery across various disease models, interestingly allowing the specific inhibition of pathogenic targets. Chloride Intracellular Channel Protein 4 (CLIC4) is a novel class of intracellular ion channel highly implicated in tumor and vascular biology. It regulates cell proliferation, apoptosis and angiogenesis; and is involved in multiple pathologic signaling pathways. Absence of specific inhibitors however impedes its advancement to translational research. Here, we integrate structural bioinformatics and experimental research approaches for the discovery and validation of small-molecule inhibitors of CLIC4. High-affinity allosteric binders were identified from a library of 1615 Food and Drug Administration (FDA)-approved drugs via a high-performance computing-powered blind-docking approach, resulting in the selection of amphotericin B and rapamycin. NMR assays confirmed the binding and conformational disruptive effects of both drugs while they also reversed stress-induced membrane translocation of CLIC4 and inhibited endothelial cell migration. Structural and dynamics simulation studies further revealed that the inhibitory mechanisms of these compounds were hinged on the allosteric modulation of the catalytic glutathione (GSH)-like site loop and the extended catalytic ß loop which may elicit interference with the catalytic activities of CLIC4. Structure-based insights from this study provide the basis for the selective targeting of CLIC4 to treat the associated pathologies.

The AtNHX1 exchanger mediates potassium compartmentation in vacuoles of transgenic tomato.

NHX-type antiporters in the tonoplast have been reported to increase the salt tolerance of various plants species, and are thought to mediate the compartmentation of Na(+) in vacuoles. However, all isoforms characterized so far catalyze both Na(+)/H(+) and K(+)/H(+) exchange. Here, we show that AtNHX1 has a critical involvement in the subcellular partitioning of K(+), which in turn affects plant K(+) nutrition and Na(+) tolerance. Transgenic tomato plants overexpressing AtNHX1 had larger K(+) vacuolar pools in all growth conditions tested, but no consistent enhancement of Na(+) accumulation was observed under salt stress. Plants overexpressing AtNHX1 have a greater capacity to retain intracellular K(+) and to withstand salt-shock. Under K(+)-limiting conditions, greater K(+) compartmentation in the vacuole occurred at the expense of the cytosolic K(+) pool, which was lower in transgenic plants. This caused the early activation of the high-affinity K(+) uptake system, enhanced K(+) uptake by roots, and increased the K(+) content in plant tissues and the xylem sap of transformed plants. Our results strongly suggest that NHX proteins are likely candidates for the H(+)-linked K(+) transport that is thought to facilitate active K(+) uptake at the tonoplast, and the partitioning of K(+) between vacuole and cytosol.

Recovery of development and functionality of nodules and plant growth in salt-stressed Pisum sativum--Rhizobium leguminosarum symbiosis by boron and calcium.

Nodules developed in Pisum sativum L. cv. Argona inoculated with Rhizobium leguminosarum bv. viciae 3841 and growing under saline conditions (75 mmol/L NaCl) are non functional and had abnormal structure. The infected cells contained a low amount of endophytic bacteria, compared to treatments without salt. Addition of B (up to 55.8 micromol/L) and Ca2+ (up to 2.72 mmol/L) increased bacterial population of host plant cells in salt-stressed nodules. Furthermore, symbiosomes developed inside the nodules from salt treated plants presented a degraded peribacteroid membrane. This effect was also prevented by combined addition of B and Ca2+. Given the importance of both nutrients in cell wall structure, the pectin fraction was studied by electron microscopy and immunological methods. Salt stress produced cells with walls dramatically altered or even degraded in several zones. Pectin polysaccharides, detected by JIM 5 monoclonal antibody, increased in cells under salinity. These effects resembled typical effects of B-deficiency reactions in cell walls, and the increase of both Ca2+ and especially B also prevented these alterations.