Vacuolar-type ATPases (V-ATPases) 1 are complex, heteromultimeric proteins consisting of a peripheral, catalytic V 1 complex and a membrane bound, ion translocating V o complex.

Isoxazolines are a novel class of parasiticides that are potent inhibitors of γ-aminobutyric acid (GABA)-gated chloride channels (GABACls) and L-glutamate-gated chloride channels (GluCls). In this study, the effects of the isoxazoline drug fluralaner on insect and acarid GABACl (RDL) and GluCl and its parasiticidal potency were investigated. We report the identification and cDNA cloning of Rhipicephalus (R.) microplus RDL and GluCl genes, and their functional expression in Xenopus laevis oocytes. The generation of six clonal HEK293 cell lines expressing Rhipicephalus microplus RDL and GluCl, Ctenocephalides felis RDL-A285 and RDL-S285, as well as Drosophila melanogaster RDLCl-A302 and RDL-S302, combined with the development of a membrane potential fluorescence dye assay allowed the comparison of ion channel inhibition by fluralaner with that of established insecticides addressing RDL and GluCl as targets. In these assays fluralaner was several orders of magnitude more potent than picrotoxinin and dieldrin, and performed 5-236 fold better than fipronil on the arthropod RDLs, while a rat GABACl remained unaffected. Comparative studies showed that R. microplus RDL is 52-fold more sensitive than R. microplus GluCl to fluralaner inhibition, confirming that the GABA-gated chloride channel is the primary target of this new parasiticide. In agreement with the superior RDL on-target activity, fluralaner outperformed dieldrin and fipronil in insecticidal screens on cat fleas (Ctenocephalides felis), yellow fever mosquito larvae (Aedes aegypti) and sheep blowfly larvae (Lucilia cuprina), as well as in acaricidal screens on cattle tick (R. microplus) adult females, brown dog tick (Rhipicephalus sanguineus) adult females and Ornithodoros moubata nymphs. These findings highlight the potential of fluralaner as a novel ectoparasiticide.

Protein kinases require strict inactivation to prevent spurious cellular signaling; overactivity can cause cancer or other diseases and necessitates selective inhibition for therapy. Rho-kinase is involved in such processes as tumor invasion, cell adhesion, smooth muscle contraction, and formation of focal adhesion fibers, as revealed using inhibitor Y-27632. Another Rho-kinase inhibitor, HA-1077 or Fasudil, is currently used in the treatment of cerebral vasospasm; the related nanomolar inhibitor H-1152P improves on its selectivity and potency. We have determined the crystal structures of HA-1077, H-1152P, and Y-27632 in complexes with protein kinase A (PKA) as a surrogate kinase to analyze Rho-kinase inhibitor binding properties. Features conserved between PKA and Rho-kinase are involved in the key binding interactions, while a combination of residues at the ATP binding pocket that are unique to Rho-kinase may explain the inhibitors' Rho-kinase selectivity. Further, a second H-1152P binding site potentially points toward PKA regulatory domain interaction modulators.

The kdpABC operon codes for the high affinity K ؉ -translocating Kdp complex (P-type ATPase) of Escherichia coli. Upon expression of this operon in minicells, a so far unrecognized small hydrophobic polypeptide, KdpF, could be identified on high resolution SDS-polyacrylamide gels in addition to the subunits KdpA, KdpB, and KdpC. Furthermore, it could be demonstrated that KdpF remains associated with the purified complex. As determined by mass spectrometry, this peptide is present in its formylated form and has a molecular mass of 3100 Da. KdpF is not essential for growth on low K ؉ (0.1 mM) medium, as shown by deletion analysis of kdpF, but proved to be indispensable for a functional enzyme complex in vitro. In the absence of KdpF, the ATPase activity of the membrane-bound Kdp complex was almost indistinguishable from that of the wild type. In contrast, the purified detergent-solubilized enzyme complex showed a dramatic decrease in enzymatic activity. However, addition of purified KdpF to the KdpABC complex restored the activity up to wild type level. It is interesting to note that the addition of high amounts of E. coli lipids had a similar effect. Although KdpF is not essential for the function of the Kdp complex in vivo, it is part of the complex and functions as a stabilizing element in vitro. The corresponding operon should now be referred to as kdpFABC.