Rescue of Mutant CFTR Trafficking Defect by the Investigational Compound MCG1516A

Although some therapeutic progress has been achieved in developing small molecules that correct F508del-CFTR defects, the mechanism of action (MoA) of these compounds remain poorly elucidated. Here, we investigated the effects and MoA of MCG1516A, a newly developed F508del-CFTR corrector. MCG1516A effects on wild-type (WT) and F508del-CFTR were assessed by immunofluorescence microscopy, and biochemical and functional assays both in cell lines and in intestinal organoids. To shed light on the MoA of MCG1516A, we evaluated its additivity to the FDA-approved corrector VX-661, low temperature, genetic revertants of F508del-CFTR (G550E, R1070W, and 4RK), and the traffic-null variant DD/AA. Finally, we explored the ability of MCG1516A to rescue trafficking and function of other CF-causing mutations. We found that MCG1516A rescues F508del-CFTR with additive effects to VX-661. A similar behavior was observed for WT-CFTR. Under low temperature incubation, F508del-CFTR demonstrated an additivity in processing and function with VX-661, but not with MCG1516A. In contrast, both compounds promoted additional effects to low temperature to WT-CFTR. MCG1516A demonstrated additivity to genetic revertant R1070W, while VX-661 was additive to G550E and 4RK. Nevertheless, none of these compounds rescued DD/AA trafficking. Both MCG1516A and VX-661 rescued CFTR processing of L206W- and R334W-CFTR with greater effects when these compounds were combined. In summary, the absence of additivity of MCG1516A to genetic revertant G550E suggests a putative binding site for this compound on NBD1:NBD2 interface. Therefore, a combination of MCG1516A with compounds able to rescue DD/AA traffic, or mimicking the actions of revertant R1070W (e.g., VX-661), could enhance correction of F508del-CFTR defects.

TRICLOSAN IS A KCNQ3 POTASSIUM CHANNEL ACTIVATOR

KCNQ channels participate in the physiology of several cell types. In neurons of the central nervous system, the primary subunits are KCNQ2, 3 and 5. Activation of these channels silence the neurons, limiting action potential duration and preventing high-frequency action potential burst. Mutation of the KCNQ genes are associated with a wide spectrum of phenotypes characterized by hyperexcitability. Activation of KCNQ channels is an attractive strategy to treat epilepsy and other hyperexcitability conditions as are the evolution of stroke and traumatic brain injury. In this work we show that triclosan, a bactericide widely used in personal care products, activates the KCNQ3 channels but not the KCNQ2. Triclosan induces a voltage shift in the activation, increases the conductance and slows the closing of the channel. The effect is independent of PIP2. The putative binding site is located in the pore region but is distinct from the binding site for retigabine. Our results indicate that triclosan is a new activator for KCNQ channels.

Comparative Molecular Dynamics Investigation of the Electromotile Hearing Protein Prestin

The mammalian protein prestin is expressed in the lateral membrane wall of the cochlear hair outer cells and is responsible for the electromotile response of the basolateral membrane, following hyperpolarisation or depolarisation of the cells. Its impairment marks the onset of severe diseases, like non-syndromic deafness. Several studies have pointed out possible key roles of residues located in the Transmembrane Domain (TMD) that differentiate mammalian prestins as incomplete transporters from the other proteins belonging to the same solute-carrier (SLC) superfamily, which are classified as complete transporters. Here, we exploit the homology of a prototypical incomplete transporter (rat prestin, rPres) and a complete transporter (zebrafish prestin, zPres) with target structures in the outward open and inward open conformations. The resulting models are then embedded in a model membrane and investigated via a rigorous molecular dynamics simulation protocol. The resulting trajectories are analyzed to obtain quantitative descriptors of the equilibration phase and to assess a structural comparison between proteins in different states, and between different proteins in the same state. Our study clearly identifies a network of key residues at the interface between the gate and the core domains of prestin that might be responsible for the conformational change observed in complete transporters and hindered in incomplete transporters. In addition, we study the pathway of Cl− ions in the presence of an applied electric field towards their putative binding site in the gate domain. Based on our simulations, we propose a tilt and shift mechanism of the helices surrounding the ion binding cavity as the working principle of the reported conformational changes in complete transporters.

LRH-1 Ameliorates Hepatic Triglyceride Accumulation via Regulation of Perilipin 5 in the Liver

Liver receptor homolog-1 (LRH-1) has emerged as a regulator of hepatic glucose, bile acid, and mitochondrial metabolism. However, the functional mechanism underlying the effect of LRH-1 on lipid mobilization has not been addressed. This study investigated the regulatory function of LRH-1 in lipid metabolism during fasting. The wild-type (WT) and LRH-1 liver-specific knockout (LKO) mice were either fed or fasted for 24 h, and the liver and serum were isolated. During fasting, the LRH-1 LKO mice showed greater accumulation of triglycerides in the liver compared to that in WT mice. Interestingly, LRH-1 LKO liver decreased the perilipin 5 (PLIN5) expression and genes involved in β-oxidation. Additionally, the LRH-1 agonist dialauroylphosphatidylcholine also enhanced PLIN5 expression in human cultured HepG2 cells. To identify new target genes of LRH-1, these findings directed to analyze the PLIN5 promoter sequence, which revealed −1620/−1614 to be a putative binding site for LRH-1. This was confirmed by promoter activity and chromatin immuno-precipitation assays. Moreover, fasted WT primary hepatocytes showed increased co-localization of PLIN5 in lipid droplets (LDs) compared to that in fasted LRH-1 LKO primary hepatocytes. Overall, these findings suggest that PLIN5 might be a novel target of LRH-1 to mobilize LDs and manage the cellular needs.

Delineation of molecular determinants for FR900359 inhibition of Gq/11 unlocks inhibition of Gαs

Heterotrimeric G proteins are essential mediators of intracellular signaling of G protein–coupled receptors. The Gq/11 subfamily consists of Gq, G11, G14, and G16 proteins, of which all but G16 are inhibited by the structurally related natural products YM-254890 and FR900359. These inhibitors act by preventing the GDP/GTP exchange, which is necessary for activation of all G proteins. A homologous putative binding site for YM-254890/FR900359 can also be found in members of the other three G protein families, Gs, Gi/o, and G12/13, but none of the published analogs of YM-254890/FR900359 have shown any inhibitory activity for any of these. To explain why the YM-254890/FR900359 scaffold only inhibits Gq/11/14, the present study delineated the molecular selectivity determinants by exchanging amino acid residues in the YM-254890/FR900359–binding site in Gq and Gs. We found that the activity of a Gs mutant with a Gq-like binding site for YM-254890/FR900359 can be inhibited by FR900359, and a minimum of three mutations are necessary to introduce inhibition in Gs. In all, this suggests that although the YM-254890/FR900359 scaffold has proven unsuccessful to derive Gs, Gi/o, and G12/13 inhibitors, the mechanism of inhibition between families of G proteins is conserved, opening up the possibility of targeting by other, novel inhibitor scaffolds. In lack of a selective Gαs inhibitor, FR900359-sensitive Gαs mutants may prove useful in studies where delicate control over Gαs signaling would be of the essence.

Ubiquitination of renal ENaC subunits in vivo

Ubiquitination of the epithelial Na+ channel (ENaC) in epithelial cells may influence trafficking and hormonal regulation of the channels. We assessed ENaC ubiquitination (ub-ENaC) in mouse and rat kidneys using affinity beads to capture ubiquitinated proteins from tissue homogenates and Western blot analysis with anti-ENaC antibodies. Ub-αENaC was observed primarily as a series of proteins of apparent molecular mass of 40–70 kDa, consistent with the addition of variable numbers of ubiquitin molecules primarily to the NH2-terminal cleaved fragment (~30 kDa) of the subunit. No significant Ub-βENaC was detected, indicating that ubiquitination of this subunit is minimal. For γENaC, the protein eluted from the affinity beads had the same apparent molecular mass as the cleaved COOH-terminal fragment of the subunit (~65 kDa). This suggests that the ubiquitinated NH2 terminus remains attached to the COOH-terminal moiety during isolation through disulfide bonds. Consistent with this, under nonreducing conditions, eluates contained material with increased molecular mass (90–150 kDa). In mice with a Liddle syndrome mutation (β566X) deleting a putative binding site for the ubiquitin ligase neural precursor cell expressed developmentally downregulated 4-2, the amount of ub-γENaC was reduced as expected. To assess aldosterone dependence of ubiquitination, we fed rats either control or low-Na+ diets for 7 days before kidney harvest. Na+ depletion increased the amounts of ub-αENaC and ub-γENaC by three- to fivefold, probably reflecting increased amounts of fully cleaved ENaC. We conclude that ubiquitination occurs after complete proteolytic processing of the subunits, contributing to retrieval and/or disposal of channels expressed at the cell surface. Diminished ubiquitination does not appear to be a major factor in aldosterone-dependent ENaC upregulation.

The Glycine Receptor Allosteric Ligands Library (GRALL)

Motivation Glycine receptors (GlyRs) mediate fast inhibitory neurotransmission in the brain and have been recognized as key pharmacological targets for pain. A large number of chemically diverse compounds that are able to modulate GlyR function both positively and negatively have been reported, which provides useful information for the development of pharmacological strategies and models for the allosteric modulation of these ion channels. Results Based on existing literature, we have collected 218 unique chemical entities with documented modulatory activities at homomeric GlyR-α1 and -α3 and built a database named GRALL. This collection includes agonists, antagonists, positive and negative allosteric modulators and a number of experimentally inactive compounds. Most importantly, for a large fraction of them a structural annotation based on their putative binding site on the receptor is provided. This type of annotation, which is currently missing in other drug banks, along with the availability of cooperativity factors from radioligand displacement experiments are expected to improve the predictivity of in silico methodologies for allosteric drug discovery and boost the development of conformation-based pharmacological approaches. Availability and implementation The GRALL library is distributed as a web-accessible database at the following link: https://ifm.chimie.unistra.fr/grall. For each molecular entry, it provides information on the chemical structure, the ligand-binding site, the direction of modulation, the potency, the 3D molecular structure and quantum-mechanical charges as determined by our in-house pipeline. Contact [email protected] Supplementary information Supplementary data are available at Bioinformatics online.

Genetic disruption of the putative binding site for Homer on DmGluRA reduces sleep in Drosophila

Homer proteins mediate plasticity and signaling at the postsynaptic density of neurons and are necessary for sleep and synaptic remodeling during sleep. The goal of this study was to investigate the mechanisms of sleep regulation by Homer signaling. Using the Drosophila animal model, we demonstrate that knockdown of Homer specifically in the brain reduces sleep and that Drosophila Homer binds to the sole Drosophila mGluR, known as DmGluRA. This is the first evidence that DmGluRA, which bears greatest homology to group II mammalian metabotropic glutamate receptors (mGluRs), shares functional homology with group I mGluRs which couple to Homer proteins in mammals. As sleep is associated with the physical dissociation of Homer and mGluRs proteins at the synapse, we sought to determine the functional necessity of Homer × DmGluRA interaction in sleep regulation. Using the CRISPR/Cas9 gene editing system, we generated a targeted amino acid replacement of the putative binding site for Homer on DmGluRA to prevent Homer and DmGluRA protein binding. We found that loss of the conserved proline-rich PPXXF sequence on DmGluRA reduces Homer/DmGluRA associations and significantly reduces sleep amount. Thus, we identify a conserved mechanism of synaptic plasticity in Drosophila and demonstrate that the interaction of Homer with DmGluRA is necessary to promote sleep.