Hydrophobic drug/toxin binding sites in voltage-dependent K+ and Na+ channels

In the Na(v)channel family the lipophilic drugs/toxins binding sites and the presence of fenestrations in the channel pore wall are well defined and categorized. No such classification exists in the much larger K(v)channel family, although certain lipophilic compounds seem to deviate from binding to well-known hydrophilic binding sites. By mapping different compound binding sites onto 3D structures of Kv channels, there appear to be three distinct lipid-exposed binding sites preserved in K(v)channels: the front and back side of the pore domain, and S2-S3/S3-S4 clefts. One or a combination of these sites is most likely the orthologous equivalent of neurotoxin site 5 in Na(v)channels. This review describes the different lipophilic binding sites and location of pore wall fenestrations within the K(v)channel family and compares it to the knowledge of Na(v)channels

Constitutive, basal, and beta-alanine-mediated activation of the human mas-related G protein-coupled receptor D induces release of the inflammatory cytokine IL-6 and is dependent on NF-kappa B signaling

G protein-coupled receptors (GPCRs) have emerged as key players in regulating (patho)physiological processes, including inflammation. Members of the Mas-related G protein coupled receptors (MRGPRs), a subfamily of GPCRs, are largely expressed by sensory neurons and known to modulate itch and pain. Several members of MRGPRs are also expressed in mast cells, macrophages, and in cardiovascular tissue, linking them to pseudo-allergic drug reactions and suggesting a pivotal role in the cardiovascular system. However, involvement of the human Mas-related G-protein coupled receptor D (MRGPRD) in the regulation of the inflammatory mediator interleukin 6 (IL-6) has not been demonstrated to date. By stimulating human MRGPRD-expressing HeLa cells with the agonist β-alanine, we observed a release of IL-6. β-alanine-induced signaling through MRGPRD was investigated further by probing downstream signaling effectors along the Gαq/Phospholipase C (PLC) pathway, which results in an IkB kinases (IKK)-mediated canonical activation of nuclear factor kappa-B (NF-κB) and stimulation of IL-6 release. This IL-6 release could be blocked by a Gαq inhibitor (YM-254890), an IKK complex inhibitor (IKK-16), and partly by a PLC inhibitor (U-73122). Additionally, we investigated the constitutive (ligand-independent) and basal activity of MRGPRD and concluded that the observed basal activity of MRGPRD is dependent on the presence of fetal bovine serum (FBS) in the culture medium. Consequently, the dynamic range for IL-6 detection as an assay for β-alanine-mediated activation of MRGPRD is substantially increased by culturing the cells in FBS free medium before treatment. Overall, the observation that MRGPRD mediates the release of IL-6 in an in vitro system, hints at a role as an inflammatory mediator and supports the notion that IL-6 can be used as a marker for MRGPRD activation in an in vitro drug screening assay

Constitutive, basal, and beta-alanine-mediated activation of the human mas-related G protein-coupled receptor D induces release of the inflammatory cytokine IL-6 and is dependent on NF-kappa B signaling

G protein-coupled receptors (GPCRs) have emerged as key players in regulating (patho)physiological processes, including inflammation. Members of the Mas-related G protein coupled receptors (MRGPRs), a subfamily of GPCRs, are largely expressed by sensory neurons and known to modulate itch and pain. Several members of MRGPRs are also expressed in mast cells, macrophages, and in cardiovascular tissue, linking them to pseudo-allergic drug reactions and suggesting a pivotal role in the cardiovascular system. However, involvement of the human Mas-related G-protein coupled receptor D (MRGPRD) in the regulation of the inflammatory mediator interleukin 6 (IL-6) has not been demonstrated to date. By stimulating human MRGPRD-expressing HeLa cells with the agonist beta-alanine, we observed a release of IL-6. beta-alanine-induced signaling through MRGPRD was investigated further by probing downstream signaling effectors along the G alpha q/Phospholipase C (PLC) pathway, which results in an IkB kinases (IKK)-mediated canonical activation of nuclear factor kappa-B (NF-kappa B) and stimulation of IL-6 release. This IL-6 release could be blocked by a G alpha q inhibitor (YM-254890), an IKK complex inhibitor (IKK-16), and partly by a PLC inhibitor (U-73122). Additionally, we investigated the constitutive (ligand-independent) and basal activity of MRGPRD and concluded that the observed basal activity of MRGPRD is dependent on the presence of fetal bovine serum (FBS) in the culture medium. Consequently, the dynamic range for IL-6 detection as an assay for beta-alanine-mediated activation of MRGPRD is substantially increased by culturing the cells in FBS free medium before treatment. Overall, the observation that MRGPRD mediates the release of IL-6 in an in vitro system, hints at a role as an inflammatory mediator and supports the notion that IL-6 can be used as a marker for MRGPRD activation in an in vitro drug screening assay

Immunosuppressive effects of new thiophene-based K▫V_V▫1.3 inhibitors

Voltage-gated potassium channel K$_V$1.3 inhibitors have been shown to be effective in preventing T-cell proliferation and activation by affecting intracellular Ca$^{2+}$ homeostasis. Here, we present the structure-activity relationship, K$_V$1.3 inhibition, and immunosuppressive effects of new thiophene-based K$_V$1.3 inhibitors with nanomolar potency on K+ current in T-lymphocytes and K$_V$1.3 inhibition on Ltk$^−$ cells. The new K$_V$1.3 inhibitor trans-18 inhibited K$_V$1.3 -mediated current in phytohemagglutinin (PHA)-activated T-lymphocytes with an IC$_{50}$ value of 26.1 nM and in mammalian Ltk− cells with an IC$_{50}$ value of 230 nM. The K$_V$1.3 inhibitor trans-18 also had nanomolar potency against K$_V$1.3 in Xenopus laevis oocytes IC$_{50}$ = 136 nM). The novel thiophene-based K$_V$1.3 inhibitors impaired intracellular Ca$^{2+}$ signaling as well as T-cell activation, proliferation, and colony formation

Design of New Potent and Selective Thiophene-Based K(V)1.3 Inhibitors and Their Potential for Anticancer Activity

The voltage-gated potassium channel KV1.3 has been recognized as a tumor marker and represents a promising new target for the discovery of new anticancer drugs. We designed a novel structural class of KV1.3 inhibitors through structural optimization of benzamide-based hit compounds and structure-activity relationship studies. The potency and selectivity of the new KV1.3 inhibitors were investigated using whole-cell patch- and voltage-clamp experiments. 2D and 3D cell models were used to determine antiproliferative activity. Structural optimization resulted in the most potent and selective KV1.3 inhibitor 44 in the series with an IC50 value of 470 nM in oocytes and 950 nM in Ltk− cells. KV1.3 inhibitor 4 induced significant apoptosis in Colo-357 spheroids, while 14, 37, 43, and 44 significantly inhibited Panc-1 proliferation