The structural insight into the functional modulation of human anion exchanger 3.

Anion exchanger 3 (AE3) is pivotal in regulating intracellular pH across excitable tissues, yet its structural intricacies and functional dynamics remain underexplored compared to other anion exchangers. This study unveils the structural insights into human AE3, including the cryo-electron microscopy structures for AE3 transmembrane domains (TMD) and a chimera combining AE3 N-terminal domain (NTD) with AE2 TMD (hAE3NTD2TMD). Our analyzes reveal a substrate binding site, an NTD-TMD interlock mechanism, and a preference for an outward-facing conformation. Unlike AE2, which has more robust acid-loading capabilities, AE3's structure, including a less stable inward-facing conformation due to missing key NTD-TMD interactions, contributes to its moderated pH-modulating activity and increased sensitivity to the inhibitor DIDS. These structural differences underline AE3's distinct functional roles in specific tissues and underscore the complex interplay between structural dynamics and functional specificity within the anion exchanger family, enhancing our understanding of the physiological and pathological roles of the anion exchanger family.

The anion exchanger slc26a3 regulates colonic mucus expansion during steady state and in response to prostaglandin E2, while Cftr regulates de novo mucus release in response to carbamylcholine.

The intestinal epithelium is covered by mucus that protects the tissue from the luminal content. Studies have shown that anion secretion via the cystic fibrosis conductance regulator (Cftr) regulates mucus formation in the small intestine. However, mechanisms regulating mucus formation in the colon are less understood. The aim of this study was to explore the role of anion transport in the regulation of mucus formation during steady state and in response to carbamylcholine (CCh) and prostaglandin E2 (PGE2). The broad-spectrum anion transport inhibitor 4,4'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS), CftrdF508 (CF) mice, and the slc26a3 inhibitor SLC26A3-IN-2 were used to inhibit anion transport. In the distal colon, steady-state mucus expansion was reduced by SLC26A3-IN-2 and normal in CF mice. PGE2 stimulated mucus expansion without de novo mucus release in wild type (WT) and CF colon via slc26a3 sensitive mechanisms, while CCh induced de novo mucus secretion in WT but not in CF colon. However, when added simultaneously, CCh and PGE2 stimulated de novo mucus secretion in the CF colon via DIDS-sensitive pathways. A similar response was observed in CF ileum that responded to CCh and PGE2 with DIDS-sensitive de novo mucus secretion. In conclusion, this study suggests that slc26a3 regulates colonic mucus expansion, while Cftr regulates CCh-induced de novo mucus secretion from ileal and distal colon crypts. Furthermore, these findings demonstrate that in the absence of a functional Cftr channel, parallel stimulation with CCh and PGE2 activates additional anion transport processes that help release mucus from intestinal goblet cells.

Synthesis and biological evaluation of DIDS analogues as efficient inhibitors of RAD51 involved in homologous recombination.

RAD51 is a pivotal protein of the homologous recombination DNA repair pathway, and is overexpressed in some cancer cells, disrupting then the efficiency of cancer-treatments. The development of RAD51 inhibitors appears as a promising solution to restore these cancer cells sensitization to radio- or chemotherapy. From a small molecule identified as a modulator of RAD51, the 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), two series of analogues with small or bulky substituents on the aromatic parts of the stilbene moiety were prepared for a structure-activity relationship study. Three compounds, the cyano analogue (12), and benzamide (23) or phenylcarbamate (29) analogues of DIDS were characterized as novel potent RAD51 inhibitors with HR inhibition in the micromolar range.

Microglial voltage-dependent anion channel 1 signaling modulates sleep deprivation-induced transition to chronic postsurgical pain.

STUDY OBJECTIVES: This study verified that sleep deprivation before and after skin/muscle incision and retraction (SMIR) surgery increased the risk of chronic pain and investigated the underlying roles of microglial voltage-dependent anion channel 1 (VDAC1) signaling. METHODS: Adult mice received 6 hours of total sleep deprivation from 1 day prior to SMIR until the third day after surgery. Mechanical and heat-evoked pain was assessed before and within 21 days after surgery. Microglial activation and changes in VDAC1 expression and oligomerization were measured. Minocycline was injected to observe the effects of inhibiting microglial activation on pain maintenance. The VDAC1 inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) and oligomerization inhibitor VBIT-4 were used to determine the roles of VDAC1 signaling on microglial adenosine 5' triphosphate (ATP) release, inflammation (IL-1ß and CCL2), and chronicity of pain. RESULTS: Sleep deprivation significantly increased the pain duration after SMIR surgery, activated microglia, and enhanced VDAC1 signaling in the spinal cord. Minocycline inhibited microglial activation and alleviated sleep deprivation-induced pain maintenance. Lipopolysaccharide (LPS)-induced microglial activation was accompanied by increased VDAC1 expression and oligomerization, and more VDAC1 was observed on the cell membrane surface compared with control. DIDS and VBIT-4 rescued LPS-induced microglial ATP release and IL-1ß and CCL2 expression. DIDS and VBIT-4 reversed sleep loss-induced microglial activation and pain chronicity in mice, similar to the effects of minocycline. No synergistic effects were found for minocycline plus VBIT-4 or DIDS. CONCLUSIONS: Perioperative sleep deprivation activated spinal microglia and increases the risk of chronic postsurgical pain in mice. VDAC1 signaling regulates microglial activation-related ATP release, inflammation, and chronicity of pain.

Upregulated ClC3 Channels/Transporters Elicit Swelling-Activated Cl- Currents and Induce Excessive Cell Proliferation in Idiopathic Pulmonary Arterial Hypertension.

Pulmonary arterial hypertension (PAH) is characterized by vascular remodeling of the pulmonary artery, which is mainly attributed to the excessive proliferation of pulmonary arterial smooth muscle cells (PASMCs) comprising the medial layer of pulmonary arteries. The activity of ion channels associated with cytosolic Ca2+ signaling regulates the pathogenesis of PAH. Limited information is currently available on the role of Cl- channels in PASMCs. Therefore, the functional expression of ClC3 channels/transporters was herein investigated in the PASMCs of normal subjects and patients with idiopathic pulmonary arterial hypertension (IPAH). Expression analyses revealed the upregulated expression of ClC3 channels/transporters at the mRNA and protein levels in IPAH-PASMCs. Hypoosmotic perfusion (230 mOsm) evoked swelling-activated Cl- currents (ICl-swell) in normal-PASMCs, whereas 100 µM 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) exerted the opposite effects. The small interfering RNA (siRNA) knockdown of ClC3 did not affect ICl-swell. On the other hand, ICl-swell was larger in IPAH-PASMCs and inhibited by DIDS and the siRNA knockdown of ClC3. IPAH-PASMCs grew more than normal-PASMCs. The growth of IPAH-PASMCs was suppressed by niflumic acid and DIDS, but not by 9-anthracenecarboxylic acid or T16Ainh-A01. The siRNA knockdown of ClC3 also inhibited the proliferation of IPAH-PASMCs. Collectively, the present results indicate that upregulated ClC3 channels/transporters are involved in ICl-swell and the excessive proliferation of IPAH-PASMCs, thereby contributing to the pathogenesis of PAH. Therefore, ClC3 channels/transporters have potential as a target of therapeutic drugs for the treatment of PAH.

Corneal dystrophy mutations R125H and R804H disable SLC4A11 by altering the extracellular pH dependence of the intracellular pK that governs H+(OH-) transport.

Mutations in the H+(OH-) conductor SLC4A11 result in corneal endothelial dystrophy. In previous studies using mouse Slc4a11, we showed that the pK value that governs the intracellular pH dependence of SLC4A11 (pKi) is influenced by extracellular pH (pHe). We also showed that some mutations result in acidic or alkaline shifts in pKi, indicating that the pH dependence of SLC4A11 is important for physiological function. An R125H mutant, located in the cytosolic amino terminus of SLC4A11, apparently causes a complete loss of function, yet the anion transport inhibitor 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS) can partially rescue SLC4A11/R125H activity. In the present study we set out to determine whether the effect of R125H is explained by an extreme shift in pKi. In Xenopus oocytes, we measured SLC4A11-mediated H+(OH-) conductance while monitoring pHi. We find that 1) the human corneal variant SLC4A11-B has a more acidic pKi than mouse Slc4a11, likely due to the presence of an NH2-terminal appendage; 2) pKi for human SLC4A11 is acid-shifted by raising pHe to 10.00; and 3) R125H and R804H mutants mediate substantial H+(OH-) conductances at pHe = 10.00, with pKi shifted into the wild-type range. These data suggest that the defect in each is a shift in pKi at physiological pHe, brought about by a disconnection in the mechanisms by which pHe influences pKi. Using de novo modeling, we show that R125 is located at the cytosolic dimer interface and suggest that this interface is critical for relaying the influence of pHe on the external face of the transmembrane domain to the intracellular, pKi-determining regions.

Erythrocyte aggregation in sudden flow arrest is linked to hyperthermia, hypoxemia, and band 3 availability.

BACKGROUND: Erythrocyte aggregation is a phenomenon that is commonly found in several pathological disease states: stroke, myocardial infarction, thermal burn injury, and COVID-19. Erythrocyte aggregation is characterized by rouleaux, closely packed stacks of cells, forming three-dimensional structures. Healthy blood flow monodisperses the red blood cells (RBCs) throughout the vasculature; however, in select pathological conditions, involving hyperthermia and hypoxemia, rouleaux formation remains and results in occlusion of microvessels with decreased perfusion. OBJECTIVES: Our objective is to address the kinetics of rouleaux formation with sudden cessation of flow in variable temperature and oxygen conditions. METHODS: RBCs used in this in vitro system were obtained from healthy human donors. Using a vertical stop-flow system aligned with a microscope, images were acquired and analyzed for increased variation in grayscale to indicate increased aggregation. The onset of aggregation after sudden cessation of flow was determined at proscribed temperatures (37-49°C) and oxygen (0%, 10%), and in the presence and absence of 4, 4'-Diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS). Both autologous and homologous plasma were tested. RESULTS: RBCs in autologous plasma aggregate faster and with a higher magnitude with both hyperthermia and hypoxemia. Preventing deoxyhemoglobin from binding to band 3 with DIDS (dissociates the cytoskeleton from the membrane) fully blocks aggregation. Further, RBC aggregation magnitude is greater in autologous plasma. CONCLUSIONS: We show that the C-terminal domain of band 3 plays a pivotal role in RBC aggregation. Further, aggregation is enhanced by hyperthermia and hypoxemia.

Overcoming T cell dysfunction in acidic pH to enhance adoptive T cell transfer immunotherapy.

The high metabolic activity and insufficient perfusion of tumors leads to the acidification of the tumor microenvironment (TME) that may inhibit the antitumor T cell activity. We found that pharmacological inhibition of the acid loader chloride/bicarbonate anion exchanger 2 (Ae2), with 4,4'-diisothiocyanatostilbene-2,2'-disulfonicacid (DIDS) enhancedCD4+ andCD8+ T cell function upon TCR activation in vitro, especially under low pH conditions. In vivo, DIDS administration delayed B16OVA tumor growth in immunocompetent mice as monotherapy or when combined with adoptive T cell transfer of OVA-specificT cells. Notably, genetic Ae2 silencing in OVA-specificT cells improvedCD4+/CD8+ T cell function in vitro as well as their antitumor activity in vivo. Similarly, genetic modification of OVA-specificT cells to overexpress Hvcn1, a selectiveH+ outward current mediator that prevents cell acidification, significantly improved T cell function in vitro, even at low pH conditions. The adoptive transfer of OVA-specificT cells overexpressing Hvcn1 exerted a better antitumor activity in B16OVA tumor-bearingmice. Hvcn1 overexpression also improved the antitumor activity of CAR T cells specific for Glypican 3 (GPC3) in mice bearing PM299L-GPC3tumors. Our results suggest that preventing intracellular acidification by regulating the expression of acidifier ion channels such as Ae2 or alkalinizer channels like Hvcn1 in tumor-specificlymphocytes enhances their antitumor response by making them more resistant to the acidic TME.

VDAC1 regulates neuronal cell loss after retinal trauma injury by a mitochondria-independent pathway.

The voltage-dependent anion channel 1 (VDAC1) was first described as a mitochondrial porin that mediates the flux of metabolites and ions, thereby integrating both cell survival and death signals. In the nervous system, the functional roles of VDAC1 remain poorly understood. Herein, the rat retina was employed to study VDAC1. First, it was observed that even subtle changes in VDAC1 levels affect neuronal survival, inducing severe alterations in the retinal morphology. We next examined the regulation of VDAC1 after traumatic retinal injury. After mechanical trauma, SOD1 translocates towards the nucleus, which is insufficient to contain the consequences of oxidative stress, as determined by the evaluation of protein carbonylation. Using in vitro models of oxidative stress and mechanical injury in primary retinal cell cultures, it was possible to determine that inhibition of VDAC1 oligomerization by 4'-diisothiocyano-2,2'-disulfonic acid stilbene (DIDS) rescues cell viability, impacting microglial cell activation. We next focused on the regulation of VDAC1 after retinal mechanical injury. VDAC1 was promptly upregulated 2 h after lesion in the plasma membrane and endoplasmic reticulum rather than in the mitochondria, and multimers of VDAC1 were assembled after lesion. DIDS intraocular application decreased apoptosis and prevented microglial polarization, which confirmed in vitro observations. Considering the role of microglia in neuroinflammation, multiplex evaluation of cytokines showed that DIDS application disorganized the inflammatory response 2 h after the lesion, matching the fast regulation of VDAC1. Taken together, data disclosed that fine regulation of VDAC1 influences neuronal survival, and pharmacological inhibition after trauma injury has neuroprotective effects. This protection may be attributed to the effects on VDAC1 abnormal accumulation in the plasma membrane, thereby controlling the activation of microglial cells. We concluded that VDAC1 is a putative therapeutic target in neuronal disorders since it integrates both death and survival cellular signaling.

Molecular Determinant of DIDS Analogs Targeting RAD51 Activity.

RAD51 is the central protein in DNA repair by homologous recombination (HR), involved in several steps of this process. It is shown that overexpression of the RAD51 protein is correlated with increased survival of cancer cells to cancer treatments. For the past decade, RAD51 overexpression-mediated resistance has justified the development of targeted inhibitors. One of the first molecules described to inhibit RAD51 was the 4,4'-diisothiocyanato-stilbene-2,2'-disulfonic acid (DIDS) molecule. This small molecule is effective in inhibiting different functions of RAD51, however its mode of action and the chemical functions involved in this inhibition have not been identified. In this work, we used several commercial molecules derived from DIDS to characterize the structural determinants involved in modulating the activity of RAD51. By combining biochemical and biophysical approaches, we have shown that DIDS and two analogs were able to inhibit the binding of RAD51 to ssDNA and prevent the formation of D-loop by RAD51. Both isothiocyanate substituents of DIDS appear to be essential in the inhibition of RAD51. These results open the way to the synthesis of new molecules derived from DIDS that should be greater modulators of RAD51 and more efficient for HR inhibition.

CO2 permeability of the rat erythrocyte membrane and its inhibition.

We have studied the CO2 permeability of the erythrocyte membrane of the rat using a mass spectrometric method that employs 18 O-labelled CO2. The method yields, in addition, the intraerythrocytic carbonic anhydrase activity and the membrane HCO3- permeability. For normal rat erythrocytes, we find at 37 °C a CO2 permeability of 0.078 ± 0.015 cm/s, an intracellular carbonic anhydrase activity of 64,100, and a bicarbonate permeability of 2.1 × 10-3 cm/s. We studied whether the rat erythrocyte membrane possesses protein CO2 channels similar to the human red cell membrane by applying the potential CO2 channel inhibitors pCMBS, Dibac, phloretin, and DIDS. Phloretin and DIDS were able to reduce the CO2 permeability by up to 50%. Since these effects cannot be attributed to the lipid part of the membrane, we conclude that the rat erythrocyte membrane is equipped with protein CO2 channels that are responsible for at least 50% of its CO2 permeability.

Characterization of a CO2-Concentrating Mechanism with Low Sodium Dependency in the Centric Diatom Chaetoceros gracilis.

Microalgae induce a CO2-concentrating mechanism (CCM) to overcome CO2-limiting stress in aquatic environments by coordinating inorganic carbon (Ci) transporters and carbonic anhydrases (CAs). Two mechanisms have been suggested to facilitate Ci uptake from aqueous media: Na+-dependent HCO3- uptake by solute carrier (SLC) family transporters and accelerated dehydration of HCO3- to CO2 by external CA in model diatoms. However, studies on ecologically and industrially important diatoms including Chaetoceros gracilis, a common food source in aquacultures, are still limited. Here, we characterized the CCM of C. gracilis using inhibitors and growth dependency on Na+ and CO2. Addition of a membrane-impermeable SLC inhibitor, 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS), or the transient removal of Na+ from the culture medium did not impair photosynthetic affinity for Ci in CO2-limiting stress conditions, but addition of a membrane-impermeable CA inhibitor, acetazolamide, decreased Ci affinity to one-third of control cultures. In culture medium containing 0.23 mM Na+ C. gracilis grew photoautotrophically by aeration with air containing 5% CO2, but not with the air containing 0.04% CO2. These results suggested that C. gracilis utilizes external CAs in its CCM to elevate photosynthetic affinity for Ci rather than plasma-membrane SLC family transporters. In addition, it is possible that low level of Na+ may support the CCM in processes other than Ci-uptake at the plasma membrane specifically in CO2-limiting conditions. Our findings provide insights into the diversity of CCMs among diatoms as well as basic information to optimize culture conditions for industrial applications.

Intestinal Absorption of Alogliptin Is Mediated by a Fruit-Juice-Sensitive Transporter.

Alogliptin (ALG), an inhibitor of dipeptidylpeptidase-4, is used in the management of type 2 diabetes mellitus, and has a high absorption rate (>60-71%), despite its low lipophilicity (logP=-1.4). Here, we aimed to clarify the mechanism of its intestinal absorption. ALG uptake into Caco-2 cells was time-, temperature-, and concentration-dependent, but was not saturated at concentrations up to 10 mmol/L. The uptake was significantly inhibited by the organic anion transporting polypeptide (OATP) substrate fexofenadine and by the OATP inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), but was not inhibited by organic cation transporter (OCT)/organic cation/carnitine transporter (OCTN) or peptide transporter 1 (PEPT1) substrates. Grapefruit, orange, and apple juices and their constituents, which are known to strongly inhibit intestinal OATPs, significantly inhibited ALG uptake into Caco-2 cells. The pH dependence was bell-shaped, indicating the involvement of a pH-sensitive transporter. However, ALG uptake by HEK293 cells overexpressing OATP2B1, a key intestinal OATP transporter of amphiphilic drugs, was not different from that of mock cells. In a rat in vivo study, apple juice reduced systemic exposure to orally administered ALG without changing the terminal half-life. These observations suggest that intestinal absorption of ALG is carrier-mediated, and involves a fruit-juice-sensitive transporter other than OATP2B1.

VDAC1 as a target in cisplatin anti-tumor activity through promoting mitochondria fusion.

Cisplatin is one of the most effective anti-cancer drugs, but its efficacy is limited by the development of resistance. Previous studies have shown that mitochondria play critical roles in cisplatin cytotoxicity, however, the exact mechanism of mitochondria involved in cisplatin sensitivity has not been clarified. In this study, cisplatin triggered mitochondrial oxidative stress and the decrease of mitochondria membrane potential in human cervical cancer cells. Then we screened a series of mitochondrial relevant inhibitors, including mitochondrial mPTP inhibitors DIDS and CsA, and mitochondrial respiratory complex inhibitors Rot and TTFA. Among these, only DIDS, as the inhibitor of mitochondrial outer membrane protein VDAC1, showed strong antagonism against cisplatin toxicity. DIDS mitigated cisplatin-induced MFN1-dependent mitochondrial fusion, mitochondrial dysfunction and oxidative damage. These findings demonstrated that VDAC1 may serve as a potential therapeutic target in the increase sensitivity of cisplatin, which provides an attractive pharmacological therapy to improve the effectiveness of chemotherapy.

The role of HCO3- in propionate-induced anion secretion across rat caecal epithelium.

Propionate, a metabolite from the microbial fermentation of carbohydrates, evokes a release of epithelial acetylcholine in rat caecum resulting in an increase of short-circuit current (Isc) in Ussing chamber experiments. The present experiments were performed in order to characterize the ionic mechanisms underlying this response which has been thought to be due to Cl- secretion. As there are regional differences within the caecal epithelium, the experiments were conducted at oral and aboral rat corpus caeci. In both caecal segments, the propionate-induced Isc (IProp) was inhibited by > 85%, when the experiments were performed either in nominally Cl-- or nominally HCO3--free buffer. In the case of Cl-, the dependency was restricted to the presence of Cl- in the serosal bath. Bumetanide, a blocker of the Na+-K+-2Cl--cotransporter, only numerically reduced IProp suggesting that a large part of this current must be carried by an ion other than Cl-. In the aboral caecum, IProp was significantly inhibited by mucosally administered stilbene derivatives (SITS, DIDS, DNDS), which block anion exchangers. Serosal Na+-free buffer reduced IProp significantly in the oral (and numerically also in aboral) corpus caeci. RT-PCR experiments revealed the expression of several forms of Na+-dependent HCO3--cotransporters in caecum, which might underlie the observed Na+ dependency. These results suggest that propionate sensing in caecum is coupled to HCO3- secretion, which functionally would stabilize luminal pH when the microbial fermentation leads to an increase in the concentration of short-chain fatty acids in the caecal lumen.

Unique Regulation of Intestinal Villus Epithelial Cl-/HCO3- Exchange by Cyclooxygenase Pathway Metabolites of Arachidonic Acid in a Mouse Model of Spontaneous Ileitis.

Electrolytes (NaCl) and fluid malabsorption cause diarrhea in inflammatory bowel disease (IBD). Coupled NaCl absorption, mediated by Na+/H+ and Cl-/HCO3- exchanges on the intestinal villus cells brush border membrane (BBM), is inhibited in IBD. Arachidonic acid metabolites (AAMs) formed via cyclooxygenase (COX) or lipoxygenase (LOX) pathways are elevated in IBD. However, their effects on NaCl absorption are not known. We treated SAMP1/YitFc (SAMP1) mice, a model of spontaneous ileitis resembling human IBD, with Arachidonyl Trifluoro Methylketone (ATMK, AAM inhibitor), or with piroxicam or MK-886, to inhibit COX or LOX pathways, respectively. Cl-/HCO3- exchange, measured as DIDS-sensitive 36Cl uptake, was significantly inhibited in villus cells and BBM vesicles of SAMP1 mice compared to AKR/J controls, an effect reversed by ATMK. Piroxicam, but not MK-886, also reversed the inhibition. Kinetic studies showed that inhibition was secondary to altered Km with no effects on Vmax. Whole cell or BBM protein levels of Down-Regulated in Adenoma (SLC26A3) and putative anion transporter-1 (SLC26A6), the two key BBM Cl-/HCO3- exchangers, were unaltered. Thus, inhibition of villus cell Cl-/HCO3- exchange by COX pathway AAMs, such as prostaglandins, via reducing the affinity of the exchanger for Cl-, and thereby causing NaCl malabsorption, could significantly contribute to IBD-associated diarrhea.

AlphaScreen Identifies MSUT2 Inhibitors for Tauopathy-Targeting Therapeutic Discovery.

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.

DIDS inhibits Vibrio vulnificus cytotoxicity by interfering with TolC-mediated RtxA1 toxin secretion.

Vibrio vulnificus (V. vulnificus) infection, frequently resulting in fatal septicemia, has become a growing health concern worldwide. The present study aimed to explore the potential agents that could protect against V. vulnificus cytotoxicity, and to analyze the possible underlying mechanisms. First, we observed that 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid disodium salt hydrate (DIDS) significantly suppressed V. vulnificus cytotoxicity to host cells by using a lactate dehydrogenase (LDH) assay. DIDS did not exhibit any effect on host cell viability, bacterial growth, microbial adhesion and swarming motility. DIDS effectively lowered V. vulnificus RtxA1 toxin-induced calcium influx into host mitochondria and RtxA1 binding to host cells. To further elucidate the underlying mechanism, the synthesis and secretion of RtxA1 toxin were investigated by Western blotting. Intriguingly, DIDS selectively inhibited the secretion of RtxA1 toxin, but did not influence its synthesis. Consequently, the outer membrane portal TolC, a key conduit for RtxA1 export coupled with tripartite efflux pumps, was examined by RT-PCR and Western blotting. We found that DIDS significantly reduced the expression of TolCV1 protein at the transcriptional level. Taken together, these results suggest that DIDS is a promising new paradigm as an antimicrobial drug that targets TolC-mediated toxin.

Voltage-gated chloride channel blocker DIDS as an acaricide for Varroa mites.

The Varroa mite is a primary driver behind periodical losses of honey bee colonies. These mites require honey bees for food and reproduction and, in turn, elicit physiological deficiencies and diseases that compromise colony health. Current acaricides for Varroa mite control, such as Apistan® (the pyrethroid tau-fluvalinate), CheckMite+® (the organophosphate coumaphos), and Apivar® (the formamidine amitraz) target the nervous system, can have adverse health effects on honey bees, and have limited effectiveness due to reported resistance issues. New target sites are needed to circumvent these obstacles in Varroa mite management, and voltage-gated chloride channels (VGCCs) are promising candidates due to their important role in the maintenance of nerve and muscle excitability in arthropod pests. Toxicological analysis of Varroa mites sensitive to tau-fluvalinate and coumaphos and Varroa mites with reduced sensitivity to these acaricides showed a significant increase in metabolic detoxification enzyme activities for the latter. Acetylcholinesterase activity in the Varroa mites exhibiting reduced mortality to coumaphos was significantly less sensitive to coumaphos-oxon compared to coumaphos-sensitive Varroa mites, which suggests target-site insensitivity to the acaricide. Voltage-gated chloride channel blocker DIDS had significantly greater field efficacy compared to Apistan® and CheckMite+® against Varroa mites from honey bee hives where tau-fluvalinate and coumaphos were observed to be ineffective, respectively. These data suggest that DIDS, and potentially other stilbene chemistries, might serve as candidates for continued field efficacy testing of alternative acaricides in apiaries where Apistan®- and CheckMite+® efficacy has been. reduced or lost for Varroa mites.

4,4'-Diisothiocyanato-2,2'-Stilbenedisulfonic Acid (DIDS) Modulates the Activity of KCNQ1/KCNE1 Channels by an Interaction with the Central Pore Region.

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.