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Tauopathies are neurological disorders characterized by intracellular tau deposits forming neurofibrillary tangles, neuropil threads, or other disease-specific aggregates composed of the protein tau. Tauopathy disorders include frontotemporal lobar degeneration, corticobasal degeneration, Pick's disease, and the largest cause of dementia, Alzheimer's disease. The lack of disease-modifying therapeutic strategies to address tauopathies remains a critical unmet need in dementia care. Thus, novel broad-spectrum tau-targeted therapeutics could have a profound impact in multiple tauopathy disorders, including Alzheimer's disease. Here we have designed a drug discovery paradigm to identify inhibitors of the pathological tau-enabling protein, MSUT2. We previously showed that activity of the RNA-binding protein MSUT2 drives tauopathy, including tau-mediated neurodegeneration and cognitive dysfunction, in mouse models. Thus, we hypothesized that MSUT2 inhibitors could be therapeutic for tauopathy disorders. Our pipeline for MSUT2 inhibitory compound identification included a primary AlphaScreen, followed by dose-response validation, a secondary fluorescence polarization orthogonal assay, a tertiary specificity screen, and a preliminary toxicity screen. Our work here serves as a proof-of-principle methodology for finding specific inhibitors of the poly(A) RNA-binding protein MSUT2 interaction. Here we identify 4,4'-diisothiocyanostilbene-2,2'-sulfonic acid (DIDS) as a potential tool compound for future work probing the mechanism of MSUT2-induced tau pathology.

Background/aims: The cardiac current IKs is carried by the KCNQ1/KCNE1-channel complex. Genetic aberrations that affect the activity of KCNQ1/KCNE1 can lead to the Long QT Syndrome 1 and 5 and, thereby, to a predisposition to sudden cardiac death. This might be prevented by pharmacological modulation of KCNQ1/KCNE1. The prototypic KCNQ1/KCNE1 activator 4,4'-Diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) represents a candidate drug. Here, we study the mechanism of DIDS action on KCNQ1/KCNE1., Methods: Channels were expressed in Xenopus oocytes and iPSC cardiomyocytes. The role of the central S6 region was investigated by alanin-screening of KCNQ1 residues 333-338. DIDS effects were measured by TEVC and MEA., Results: DIDS-action is influenced by the presence of KCNE1 but not by KCNQ1/KCNE1 stochiometry. V334A produces a significant higher increase in current amplitude, whereas deactivation (slowdown) DIDS-sensitivity is affected by residues 334-338., Conclusion: We show that the central S6 region serves as a hub for allosteric channel activation by the drug and that DIDS shortens the pseudo QT interval in iPSC cardiomyocytes. The elucidation of the structural and mechanistic underpinnings of the DIDS action on KCNQ1/KCNE1 might allow for a targeted design of DIDS derivatives with improved potency and selectivity., Competing Interests: The authors declare no conflict of interest exist., (© Copyright by the Author(s). Published by Cell Physiol Biochem Press.)

MitoNEET is an outer mitochondrial membrane protein essential for sensing and regulation of iron and reactive oxygen species (ROS) homeostasis. It is a key player in multiple human maladies including diabetes, cancer, neurodegeneration, and Parkinson's diseases. In healthy cells, mitoNEET receives its clusters from the mitochondrion and transfers them to acceptor proteins in a process that could be altered by drugs or during illness. Here, we report that mitoNEET regulates the outer-mitochondrial membrane (OMM) protein voltage-dependent anion channel 1 (VDAC1). VDAC1 is a crucial player in the cross talk between the mitochondria and the cytosol. VDAC proteins function to regulate metabolites, ions, ROS, and fatty acid transport, as well as function as a "governator" sentry for the transport of metabolites and ions between the cytosol and the mitochondria. We find that the redox-sensitive [2Fe-2S] cluster protein mitoNEET gates VDAC1 when mitoNEET is oxidized. Addition of the VDAC inhibitor 4,4'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS) prevents both mitoNEET binding in vitro and mitoNEET-dependent mitochondrial iron accumulation in situ. We find that the DIDS inhibitor does not alter the redox state of MitoNEET. Taken together, our data indicate that mitoNEET regulates VDAC in a redox-dependent manner in cells, closing the pore and likely disrupting VDAC's flow of metabolites., Competing Interests: The authors declare no conflict of interest., (Copyright © 2019 the Author(s). Published by PNAS.)

Background/aims: Pendrin is a Cl-/I-/HCO3- exchanger playing a fundamental role in controlling blood pressure and airway function, therefore representing an attractive target for the treatment of hypertensive states and respiratory distresses. A review of the literature regarding the ability of some compounds (namely several known inhibitors of ion transport) to block pendrin activity revealed discordant findings. These incongruous findings may be due, in part, to the concentration of compound and/or the nature of the model system used in the study., Methods: Pendrin activity was evaluated by measuring pendrin-dependent iodide influx following overexpression of the transporter in a human kidney cell line, in the presence of selected test compounds or the respective vehicles., Results: Pendrin activity was significantly hampered by 0.1 mM 5-nitro-2-[(3-phenylpropyl)amino]benzoic acid (NPPB), niflumic acid and tenidap, but was resistant to 0.1 mM 4, 4'-diisothiocyano-2, 2'-stilbene-disulfonic acid (DIDS), furosemide and probenecid., Conclusions: The results of the present study indicate that clinically effective non-steroidal anti-inflammatory drugs (niflumic acid and tenidap) directly inhibit pendrin activity., (© 2016 The Author(s) Published by S. Karger AG, Basel.)

The cellular DNA repair hRAD51 protein has been shown to restrict HIV-1 integration both in vitro and in vivo. To investigate its regulatory functions, we performed a pharmacological analysis of the retroviral integration modulation by hRAD51. We found that, in vitro, chemical activation of hRAD51 stimulates its integration inhibitory properties, whereas inhibition of hRAD51 decreases the integration restriction, indicating that the modulation of HIV-1 integration depends on the hRAD51 recombinase activity. Cellular analyses demonstrated that cells exhibiting high hRAD51 levels prior to de novo infection are more resistant to integration. On the other hand, when hRAD51 was activated during integration, cells were more permissive. Altogether, these data establish the functional link between hRAD51 activity and HIV-1 integration. Our results highlight the multiple and opposite effects of the recombinase during integration and provide new insights into the cellular regulation of HIV-1 replication., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Mutations in the gene-encoding cystic fibrosis transmembrane conductance regulator (CFTR) cause defective transepithelial transport of chloride (Cl(-)) ions and fluid, thereby becoming responsible for the onset of cystic fibrosis (CF). One strategy to reduce the pathophysiology associated with CF is to increase Cl(-) transport through alternative pathways. In this paper, we demonstrate that a small synthetic molecule which forms Cl(-) channels to mediate Cl(-) transport across lipid bilayer membranes is capable of restoring Cl(-) permeability in human CF epithelial cells; as a result, it has the potential to become a lead compound for the treatment of human diseases associated with Cl(-) channel dysfunction.

Fatty acids flow from adipocytes to nonadipose tissues during fasting and exercise and normally are fully oxidized. To determine if nonadipose tissues can export unoxidized FA when FA influx exceeds oxidation, neonatal cardiomyocytes were cultured in 1 microCi (14)C-palmitate in the presence of etomoxir to block oxidation. The cells took up and stored 25% of the radioactivity as (14)C-triacylglycerol in 12 h, but 4.5% of the label was released in 3 h and comigrated with (14)C-palmitate. Both uptake and release of radioactivity were increased by insulin and reduced by the nonspecific inhibitors of FA transporters phloretin and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS). Perfused hearts from etomoxir-treated lean rats released 221 +/- 59 nmol/10 min of FA. Hearts from high-fat-fed lean rats released 366 +/- 172 nmol/10 min (P < 0.05). Hearts from obese rats released 744 +/- 260 and 1,578 +/- 630 nmol/10 min at 8 and 12 weeks of age, respectively. Perfusion with insulin increased FA release by 32%. In vitro and ex vivo findings suggest that nonadipose tissues such as myocardium can export FA when the unoxidized lipid content is excessive.

The highly homologous Cl(-) channels CLC-Ka and CLC-Kb are important for water and salt conservation in the kidney and for the production of endolymph in the inner ear. Mutations in CLC-Kb lead to Bartter's syndrome and mutations in the small CLC-K subunit barttin lead to Bartter's syndrome and deafness. Here we show that CLC-Ka is blocked by the recently identified blocker 2-(p-chlorophenoxy)-3-phenylpropionic acid of the rat channel CLC-K1 with an apparent K(D) approximately 80 microM. We also found that DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid), a generic Cl(-) channel blocker, inhibits CLC-Ka (K(D) approximately 90 microM). Surprisingly, the highly homologous channel CLC-Kb is fivefold to sixfold less sensitive to both compounds. Guided by the crystal structure of bacterial CLC proteins, we identify two amino acids, N68/D68 and G72/E72, in CLC-Ka and CLC-Kb, respectively, that are responsible for the differential drug sensitivity. Both residues expose their side chains in the extracellular pore mouth, delineating the probable drug binding site. These novel CLC-K channel blockers are promising lead compounds for the development of new diuretic drugs.

Band 3 Memphis variant II is a mutant anion-exchange protein associated with the Diego a+ blood group antigen. There are two mutations in this transporter: Lys-56-->Glu within the cytoplasmic domain, and Pro-854-->Leu within the membrane-bound domain. The Pro-854 mutation, which is thought to give rise to the antigenicity, is located within the C-terminal subdomain of the membrane-bound domain. Yet, there is an apparent enhancement in the rate of covalent binding of H2DIDS (4,4'-di-isothiocyanatodihydro-2, 2'-stilbenedisulphonate) to 'lysine A' (Lys-539) in the N-terminal subdomain, suggesting widespread conformational changes. In this report, we have used various kinetic assays which differentiate between conformational changes in the two subdomains, to characterize the stilbenedisulphonate site on band 3 Memphis variant II. We have found a significantly higher H2DIDS (a C-terminal-sensitive inhibitor) affinity for band 3 Memphis variant II, due to a lower H2DIDS 'off' rate constant, but no difference was found between mutant and control when DBDS (4,4'-dibenzamido-2,2'-stilbenedisulphonate) (a C-terminal-insensitive inhibitor) 'off' rates were measured. Furthermore, there were no differences in the rates of covalent binding to lysine A, for either DIDS (4,4'-di-isothiocyanato-2,2'-stilbenedisulphonate) or H2DIDS. However, the rate of covalent intrasubunit cross-linking of Lys-539 and Lys-851 by H2DIDS was abnormally low for band 3 Memphis variant II. These results suggest that the Pro-854-->Leu mutation causes a localized conformational change in the C-terminal subdomain of band 3.

1. Using a grease-gap recording technique we have investigated the effects of some antagonists of P2-purinoceptors on the depolarization of the rat isolated superior cervical ganglion evoked by 100 microM alpha, beta-methylene-adenosine 5'-triphosphate (alpha,beta-MeATP) or uridine 5'-triphosphate (UTP). The effects of the putative P2Z-purinoceptor antagonist, coomassie brilliant blue G, putative P2X-purinoceptor antagonist, 4,4'-diisothiocyanatostilbene-2,2'-disulphonate (DIDS) and uniblue A (an analogue of the P2Y- and P2X-purinoceptor antagonist reactive blue 2) were investigated. 2. At the highest concentration examined uniblue A (300 microM) depressed alpha,beta-MeATP-induced depolarization and at 100 and 300 microM enhanced UTP-evoked depolarizations. Coomassie brilliant blue G (1 and 10 microM) did not affect depolarizations evoked by alpha,beta-MeATP or UTP. Depolarizations evoked by potassium (5 mM) or muscarine (100 nM) were unaltered by either coomassie brilliant blue G or uniblue A. Uniblue A (100 and 300 microM) produced a concentration-dependent depression of hyperpolarizations evoked by adenosine (100 microM) whereas coomassie brilliant blue G at up to 10 microM, did not alter adenosine-induced hyperpolarizations. 3. DIDS (30 and 100 microM) did not alter adenosine-evoked hyperpolarizations, or depolarizations evoked by potassium or UTP. DIDS at 100 microM did not alter depolarizations evoked by muscarine. In contrast DIDS produced a concentration-dependent depression of alpha,beta-MeATP-evoked depolarizations. 4. These results are consistent with the proposal that uniblue A and DIDS but not coomassie brilliant blue G are antagonists of P2-purinoceptors and that uniblue A is also an antagonist at P1-purinoceptors present on the rat superior cervical ganglion. 5. The ability of uniblue A and DIDS to distinguish between depolarizations evoked by UTP and alpha,beta-MeATP provides further justification for the proposal that these nucleotides activate separate receptors present on the rat superior cervical ganglion, i.e. pyrimidinoceptors and P2-purinoceptors respectively.