Photodegradation of anthelmintic drugs under natural sunlight and simulated irradiation: kinetics, mechanisms, transformation products, and toxicity.

Albendazole (ALB) and bithionol (BIT) are two anthelmintic drugs (ADs) with high consumption from benzimidazole group and diphenylsulfide group, respectively. However, information on the transformation of the two anthelmintics under environmental condition is scare. Therefore, in the present study, we investigated the natural attenuation of the two ADs in the aquatic environment, including biodegradation, hydrolysis, and direct and indirect photodegradation. The direct photodegradation occupied a vast portion among other degradation pathways of the two ADs in natural water, with near-surface summer half-lives of 0.272-0.387 h and 0.110-0.520 h for ALB and BIT, respectively. Suspended particles in water were found to facilitate the photodegradation of the two ADs. Study on the indirect photodegradation demonstrated the positive roles of singlet oxygen (1O2) and excited triplet dissolved organic matter (3DOM*) in the photolysis of the two ADs, whereas the hydroxyl radical (•OH) affected little on the overall photodegradation procedures of ALB due to the scavenging effect of HCO3-. Dual effects of DO, DOM, HCO3-, NO3-, and NO2- on the photodegradation of ALB and BIT were perceived. Transformation intermediates (TIs) of the two ADs during photodegradation were analyzed by UHPLC-QTOF-MS. Six TIs of ALB were identified, including a broad-spectrum fungicide carbendazim and another common AD ricobendazole. Two TIs of BIT yielded from dechlorination were also detected. Probable transformation mechanism and predicted aquatic ecotoxicity based on the identified TIs were unveiled.

Veterinary deworming agent-induced toxic optic neuropathy: a case report.

BACKGROUND: Veterinary antiparasitic drugs are widely used in countries and regions in which parasitic diseases are endemic, which leads to the risk of accidental ingestion and poisoning in humans.  CASE PRESENTATION: A 40-year-old male patient with a history of cirrhosis sought medical attention on November 25, 2021, due to progressive vision loss. He had previously taken triclabendazole and bithionol and was diagnosed with toxic optic neuropathy on examination. Steroid, neurotonic, and high-pressure oxygen therapy were ineffective. CONCLUSIONS: Triclabendazole and bithionol have potential risk of optic neurotoxicity and should be considered for enhanced supervision and warning labels.

Bithionol Restores Sensitivity of Multidrug-Resistant Gram-Negative Bacteria to Colistin with Antimicrobial and Anti-biofilm Effects.

Being among the few last-resort antibiotics, colistin (COL) has been used to treat severe infectious diseases, such as those caused by multidrug-resistant Gram-negative bacteria (MDR GNB). However, the appearance of colistin-resistant (COL-R) GNB has been frequently reported. Therefore, novel antimicrobial strategies need to be urgently sought to address this resistance challenge. In the present study, antimicrobial drug screening conducted revealed that bithionol (BT), approved by the Food and Drug Administration and used as an anthelminthic drug for paragonimiasis, exhibited a synergistic antibacterial effect with COL. Clinically isolated COL-R GNB were used as candidates to evaluate the synergistic antibacterial activity. The results revealed that BT could significantly reverse the sensitivity of COL-R GNB to COL. Furthermore, the combined application of BT and COL can reduce bacterial biofilm formation and have a scavenging effect on the mature biofilm in vitro. The damage caused to the bacterial cell membrane integrity by the BT/COL combination was observed under a fluorescence microscope. The fluorescence intensity of reactive oxygen species also increased in the experimental group. The BT/COL combination also exhibited a synergistic antibacterial effect in vivo. Importantly, BT was confirmed to be safe at the highest concentrations that exerted synergistic effects on all tested strains. In conclusion, our findings demonstrated that BT exerted synergistic antimicrobial and anti-biofilm effects when combined with COL against MDR organisms, especially COL-R GNB, in vitro and in vivo. The findings thus provide a reference for the clinical response to the serious challenge of MDR GNB and the exploitation of the potential antibacterial activities of existing clinical non-antibacterial drugs.

The potential target of bithionol against Staphylococcus aureus: design, synthesis and application of biotinylated probes Bio-A2.

This study aims to explore the potential targets of bithionol in Staphylococcus aureus.The four bithionol biotinylated probes Bio-A2-1, Bio-A2-2, Bio-A2-3, and Bio-A2-4 were synthesized, the minimal inhibitory concentrations (MICs) of these probes against S. aureus were determined. The bithionol binding proteins in S. aureus were identified through immunoprecipitation and LC-MS/MS with bithionol biotinylated probe. The biotinylated bithionol probes Bio-A2-1 and Bio-A2-3 displayed antibacterial activities against S. aureus. The Bio-A2-1 showed lower MICs than Bio-A2-3, and both with the MIC50/MIC90 at 12.5/12.5 µM against S. aureus clinical isolates. The inhibition rates of bithionol biotinylated probes Bio-A2-1 and Bio-A2-3 on the biofilm formation of S. aureus were comparable to that of bithionol, and were stronger than that of Bio-A2-2 and Bio-A2-4. The biofilm formation of 10 out of 12S. aureus clinical isolates could be inhibited by Bio-A2-1 (at 1/4×, or 1/2× MICs). There are three proteins identified in S. aureus through immunoprecipitation and LC-MS/MS with bithionol biotinylated probe Bio-A2-1: Protein translocase subunit SecA 1 (secA1), Alanine--tRNA ligase (alaS) and DNA gyrase subunit A (gyrA), and in which the SecA1 protein the highest coverage and the most unique peptides. The LYS112, GLN143, ASP213, GLY496 and ASP498 of SecA1 protein act as hydrogen acceptors to form 6 hydrogen bonds with bithionol biotinylated probe Bio-A2-1 by molecular docking analysis. In conclusion, the bithionol biotinylated probe Bio-A2-1 has antibacterial and anti-biofilm activities against S. aureus, and SecA1 was probably one of the potential targets of bithionol in S. aureus.

False-negative chemicals in ESR-based photosafety test (ESR-PT) and their significance for photosafety evaluations: examples of bithionol, fenticlor and cilnidipine.

The electron spin resonance (ESR)-based photosafety test (ESR-PT) is a non-animal prediction test for photosafety evaluations that can be used even for hydrophobic chemicals; the method is based on the detection of singlet oxygen generation using ESR spectroscopy and showing high accuracy for compounds with known photosafety information. During the process of extending the application data for ESR-PT, we found three false-negative chemicals: bithionol, fenticlor and cilnidipine. These chemicals did not show the characteristic triplet signal of 4-hydroxy-2,2,6,6-tetramethyl-piperidine-1-oxyl (4-hydroxy-TEMPO), which is used as a classifier for positive or negative chemicals; instead, bithionol and fenticlor induced an apparent single peak signal with a g-value of 2.0048, while cilnidipine produced a small, fragmented signal. Bithionol and fenticlor reportedly induce free radicals, and positive phototoxic or photoallergic evidence have been reported. Although the small, fragmented signal observed for cilnidipine was confirmed to be identical to that of a phenylnitroxy radical by the computer simulation, the significance of this chemical for photosafety considerations may be low because cilnidipine has quite a low incidence of phototoxic or photoallergic reactions in humans. Accordingly, the current ESR-PT protocol should be improved to detect free radical photoproducts generated from chemicals such as bithionol and fenticlor, thereby helping to reduce false negatives in ESR-PT.

Discovery and Mechanistic Study of Mycobacterium tuberculosis PafA Inhibitors.

Tuberculosis is caused by the bacterium Mycobacterium tuberculosis (Mtb) and is ranked as the second killer infectious disease after COVID-19. Proteasome accessory factor A (PafA) is considered an attractive target because of its low sequence conservation in humans and its role in virulence. In this study, we designed a mutant of Mtb PafA that enabled large-scale purification of active PafA. Using a devised high-throughput screening assay, two PafA inhibitors were discovered. ST1926 inhibited Mtb PafA by binding in the Pup binding groove, but it was less active against Corynebacterium glutamicum PafA because the ST1926-binding residues are not conserved. Bithionol bound to the conserved ATP-binding pocket, thereby, inhibits PafA in an ATP-competitive manner. Both ST1926 and bithionol inhibited the growth of an attenuated Mtb strain (H37Ra) at micromolar concentrations. Our work thus provides new tools for tuberculosis research and a foundation for future PafA-targeted drug development for treating tuberculosis.

Repositioning of the Anthelmintic Drugs Bithionol and Triclabendazole as Transthyretin Amyloidogenesis Inhibitors.

Transthyretin (TTR) is a causative protein of TTR amyloidosis (ATTR amyloidosis), a general term for diseases characterized by deposition of TTR amyloid fibrils in specific organs. ATTR amyloidosis can be ameliorated by stabilization of the TTR tetramer through the binding of small molecules. Here, we show that the clinical anthelmintic drugs bithionol (42) and triclabendazole (43) potently inhibit aggregation of the amyloidogenic variant V30M-TTR. A competitive binding assay using a fluorescence probe showed that the binding affinity of 42 with V30M-TTR was significantly higher than that of the first-in-class drug tafamidis (1), and the binding affinity of 43 was similar to that of 1. The crystallographic and thermodynamic analysis revealed that 42 efficiently occupied the halogen-binding grooves of TTR, resulting in the favorable binding entropy. Multifaceted in vitro studies of anthelmintic drugs have the potential to reposition these drugs as ATTR amyloidosis inhibitors.

Biosynthesis of N-Docosahexanoylethanolamine from Unesterified Docosahexaenoic Acid and Docosahexaenoyl-Lysophosphatidylcholine in Neuronal Cells.

We investigated the synthesis of N-docosahexaenoylethanolamine (synaptamide) in neuronal cells from unesterified docosahexaenoic acid (DHA) or DHA-lysophosphatidylcholine (DHA-lysoPC), the two major lipid forms that deliver DHA to the brain, in order to understand the formation of this neurotrophic and neuroprotective metabolite of DHA in the brain. Both substrates were taken up in Neuro2A cells and metabolized to N-docosahexaenoylphosphatidylethanolamine (NDoPE) and synaptamide in a time- and concentration-dependent manner, but unesterified DHA was 1.5 to 2.4 times more effective than DHA-lysoPC at equimolar concentrations. The plasmalogen NDoPE (pNDoPE) amounted more than 80% of NDoPE produced from DHA or DHA-lysoPC, with 16-carbon-pNDoPE being the most abundant species. Inhibition of N-acylphosphatidylethanolamine-phospholipase D (NAPE-PLD) by hexachlorophene or bithionol significantly decreased the synaptamide production, indicating that synaptamide synthesis is mediated at least in part via NDoPE hydrolysis. NDoPE formation occurred much more rapidly than synaptamide production, indicating a precursor-product relationship. Although NDoPE is an intermediate for synaptamide biosynthesis, only about 1% of newly synthesized NDoPE was converted to synaptamide, possibly suggesting additional biological function of NDoPE, particularly for pNDoPE, which is the major form of NDoPE produced.

Symmetrically substituted dichlorophenes inhibit N-acyl-phosphatidylethanolamine phospholipase D.

N-Acyl-phosphatidylethanolamine phospholipase D (NAPE-PLD) (EC 3.1.4.4) catalyzes the final step in the biosynthesis of N-acyl-ethanolamides. Reduced NAPE-PLD expression and activity may contribute to obesity and inflammation, but a lack of effective NAPE-PLD inhibitors has been a major obstacle to elucidating the role of NAPE-PLD and N-acyl-ethanolamide biosynthesis in these processes. The endogenous bile acid lithocholic acid (LCA) inhibits NAPE-PLD activity (with an IC50 of 68 µm), but LCA is also a highly potent ligand for TGR5 (EC50 0.52 µm). Recently, the first selective small-molecule inhibitor of NAPE-PLD, ARN19874, has been reported (having an IC50 of 34 µm). To identify more potent inhibitors of NAPE-PLD, here we used a quenched fluorescent NAPE analog, PED-A1, as a substrate for recombinant mouse Nape-pld to screen a panel of bile acids and a library of experimental compounds (the Spectrum Collection). Muricholic acids and several other bile acids inhibited Nape-pld with potency similar to that of LCA. We identified 14 potent Nape-pld inhibitors in the Spectrum Collection, with the two most potent (IC50 = ∼2 µm) being symmetrically substituted dichlorophenes, i.e. hexachlorophene and bithionol. Structure-activity relationship assays using additional substituted dichlorophenes identified key moieties needed for Nape-pld inhibition. Both hexachlorophene and bithionol exhibited significant selectivity for Nape-pld compared with nontarget lipase activities such as Streptomyces chromofuscus PLD or serum lipase. Both also effectively inhibited NAPE-PLD activity in cultured HEK293 cells. We conclude that symmetrically substituted dichlorophenes potently inhibit NAPE-PLD in cultured cells and have significant selectivity for NAPE-PLD versus other tissue-associated lipases.

Addition of ethylamines to the phenols of bithionol and synthetic retinoids does not elicit activity in gram-negative bacteria.

Our labs have demonstrated the activity of bithionol and synthetic retinoids against methicillin-resistant Staphylococcus aureus (MRSA), as well as their membrane-acting mechanism of action. However, the compounds lack activity in gram-negative species. Herein, we apply a known strategy for converting gram-positive agents into broad-spectrum therapies: addition of an alkylamine. By appending an alkylamine to the phenols of these known membrane disruptors, we test whether this approach is applicable to our compounds. Ultimately, biological testing in four MRSA strains and three gram-negative species showed abolished or diminished activity in all our analogs compared to their parent compounds and no gram-negative activity. Thus, we find that alkylamines would not elicit broad-spectrum activity from bithionol or CD437 derivatives.

Dissecting the Antenna in Human Glutamate Dehydrogenase: Understanding Its Role in Subunit Communication and Allosteric Regulation.

Glutamate dehydrogenase (GDH) is a homohexameric enzyme that catalyzes the reversible oxidative deamination of l-glutamate. While GDH is found in all living organisms, only that from animals is highly allosterically regulated by a wide array of metabolites. Because only animal GDH has a 50-residue antenna domain, we hypothesized that it was critical for allostery. To this end, we previously replaced the antenna with the loop found in bacteria, and the resulting chimera was no longer regulated by purine nucleotides. Hence, it seemed logical that the purpose of the antenna is to exert the subunit communication necessary for heterotrophic allosteric regulation. Here, we revisit the antenna deletion studies by retaining 10 more of the human GDH (hGDH) residues without adding the bacterial loop. Unexpectedly, the results were profoundly different than before. The basal activity of the mutant is only ∼13% of that of the wild type but ∼100 times more sensitive to all allosteric activators. In contrast, the mutant is still affected by all of the tested inhibitors to approximately the same degree. The resulting antenna-less mutant retained its negative cooperativity with respect to the coenzyme, again suggesting that intersubunit communication is intact. Finally, the mutant still exhibits substrate inhibition, albeit there are differences in the details. We present a model in which the majority of the antenna is not directly involved in allosteric regulation per se but rather may be responsible for improving enzymatic efficiency by acting as a conduit for substrate binding energy between subunits.

A selective membrane-targeting repurposed antibiotic with activity against persistent methicillin-resistant Staphylococcus aureus.

Treatment of Staphylococcus aureus infections is complicated by the development of antibiotic tolerance, a consequence of the ability of S. aureus to enter into a nongrowing, dormant state in which the organisms are referred to as persisters. We report that the clinically approved anthelmintic agent bithionol kills methicillin-resistant S. aureus (MRSA) persister cells, which correlates with its ability to disrupt the integrity of Gram-positive bacterial membranes. Critically, bithionol exhibits significant selectivity for bacterial compared with mammalian cell membranes. All-atom molecular dynamics (MD) simulations demonstrate that the selectivity of bithionol for bacterial membranes correlates with its ability to penetrate and embed in bacterial-mimic lipid bilayers, but not in cholesterol-rich mammalian-mimic lipid bilayers. In addition to causing rapid membrane permeabilization, the insertion of bithionol increases membrane fluidity. By using bithionol and nTZDpa (another membrane-active antimicrobial agent), as well as analogs of these compounds, we show that the activity of membrane-active compounds against MRSA persisters positively correlates with their ability to increase membrane fluidity, thereby establishing an accurate biophysical indicator for estimating antipersister potency. Finally, we demonstrate that, in combination with gentamicin, bithionol effectively reduces bacterial burdens in a mouse model of chronic deep-seated MRSA infection. This work highlights the potential repurposing of bithionol as an antipersister therapeutic agent.

Sorption and leaching behavior of bithionol and levamisole in soils.

The batch experiments were conducted to understand sorption process of bithionol (BIT) in yellow soil (YS) and red soil (RS), while column leaching experiments were performed to evaluate the leaching behavior of BIT and levamisole (LEV) in the tested soils. The adsorption and desorption data fitted well with the Freundlich isotherms (R2 ≥ 0.94). The distribution coefficient of BIT in the YS and RS were 104 and 98.3 L/kg, respectively. Hysteresis was observed for bithionol desorption in the YS and RS, with hysteresis coefficient of 0.917 and 0.928, respectively. Dissolved organic matter (DOM) addition and acid condition enhanced the adsorption of BIT in the soil. Both BIT and LEV showed poor leaching potential in the tested soils. More than 80% of BIT and LEV remained in the surface soil layer and the amount of the two target compounds in the leachates accounted for less than 1% of overall recovery. DOM showed little influence on the concentration of BIT and LEV in the leachates collected at different time. The results could fill the gap on the behavior of BIT and LEV in soil under laboratory conditions.

The inhibitors of soluble adenylate cyclase 2-OHE, KH7, and bithionol compromise mitochondrial ATP production by distinct mechanisms.

Soluble adenylate cyclase (sAC) is a non-plasma membrane-bound isoform of the adenylate cyclases signaling via the canonical second messenger, 3',5'-cyclic AMP (cAMP). sAC is involved in key physiological processes such as insulin release, sperm motility, and energy metabolism. Thus, sAC has attracted interest as a putative drug target and attempts have been made to develop selective inhibitors. Since sAC has a binding constant for its substrate, ATP, in the millimolar range, reductions in mitochondrial ATP production may be part of the mechanism-of-action of sAC inhibitors and the potential of these compounds to study the physiological outcomes of inhibition of sAC might be severely hampered by this. Here, we evaluate the effects of two commonly employed inhibitors, 2-OHE and KH7, on mitochondrial ATP production and energy metabolism. For comparison, we included a recently identified inhibitor of sAC, bithionol. Employing mitochondria isolated from mouse brain, we show that all three compounds are able to curb ATP production albeit via distinct mechanisms. Bithionol and KH7 mainly inhibit ATP production by working as a classical uncoupler whereas 2-OHE mainly works by decreasing mitochondrial respiration. These findings were corroborated by investigating energy metabolism in acute brain slices from mice. Since all three sAC inhibitors are shown to curb mitochondrial ATP production and affect energy metabolism, caution should be exercised when employed to study the physiological roles of sAC or for validating sAC as a drug target.

Evaluation of the cytotoxicity of the Bithionol-paclitaxel combination in a panel of human ovarian cancer cell lines.

Previously, Bithionol (BT) was shown to enhance the chemosensitivity of ovarian cancer cell lines to cisplatin treatment. In the present study, we focused on the anti-tumor potential of the BT-paclitaxel combination when added to a panel of ovarian cancer cell lines. This in vitro study aimed to 1) determine the optimum schedule for combination of BT and paclitaxel and 2) assess the nature and mechanism(s) underlying BT-paclitaxel interactions. The cytotoxic effects of both drugs either alone or in combination were assessed by presto-blue cell viability assay using six human ovarian cancer cell lines. Inhibitory concentrations to achieve 50% cell death (IC50) were determined for BT and paclitaxel in each cell line. Changes in levels of cleaved PARP, XIAP, bcl-2, bcl-xL, p21 and p27 were determined via immunoblot. Luminescent and colorimetric assays were used to determine caspases 3/7 and autotaxin (ATX) activity. Cellular reactive oxygen species (ROS) were measured by flow cytometry. Our results show that the efficacy of the BT-paclitaxel combination depends upon the concentrations and sequence of addition of paclitaxel and BT. Pretreatment with BT followed by paclitaxel resulted in antagonistic interactions whereas synergistic interactions were observed when both drugs were added simultaneously or when cells were pretreated with paclitaxel followed by BT. Synergistic interactions between BT and paclitaxel were attributed to increased ROS generation and enhanced apoptosis. Decreased expression of pro-survival factors (XIAP, bcl-2, bcl-xL) and increased expression of pro-apoptotic factors (caspases 3/7, PARP cleavage) was observed. Additionally, increased expression of key cell cycle regulators p21 and p27 was observed. These results show that BT and paclitaxel interacted synergistically at most drug ratios which, however, was highly dependent on the sequence of the addition of drugs. Our results suggest that BT-paclitaxel combination therapy may be effective in sensitizing ovarian cancer cells to paclitaxel treatment, thus mitigating some of the toxic effects associated with high doses of paclitaxel.

Bithionol residue analysis in animal-derived food products by an effective and rugged extraction method coupled with liquid chromatography-tandem mass spectrometry.

Herein, we developed a simple analytical procedure for the quantitation of bithionol residues in animal-derived food products such as porcine muscle, eggs, milk, eel, flatfish, and shrimp using a modified quick, easy, cheap, effective, rugged, and safe (QuEChERS) extraction method coupled with liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI+/MS-MS). Samples were extracted with 0.1% solution of formic acid in acetonitrile and the extract was purified using a C18 sorbent. Separation was performed on a Waters XBridge™ C18 reversed-phase analytical column using 0.1% solution of formic acid/acetonitrile as the mobile phase. Six-point matrix-matched calibration indicated good linearity, with the calculated coefficients of determination (R2) being≥0.9813. Intra- and inter-day recoveries (determined at spiking levels equivalent to 1×and 2×the limit of quantitation (0.25µg/kg)) ranged between 80.0 and 94.0%, with the corresponding relative standard deviations (RSDs) being≤8.2%. The developed experimental protocol was applied to different samples purchased from local markets in Seoul, which were tested negative for bithionol residues. In conclusion, the proposed method proved to be versatile and precise, being ideally suited for the routine detection of bithionol residues in animal-derived food products with various protein and fat contents.

Evaluation of the cytotoxicity of the Bithionol - cisplatin combination in a panel of human ovarian cancer cell lines.

BACKGROUND: Combination drug therapy appears a promising approach to overcome drug resistance and reduce drug-related toxicities in ovarian cancer treatments. In this in vitro study, we evaluated the antitumor efficacy of cisplatin in combination with Bithionol (BT) against a panel of ovarian cancer cell lines with special focus on cisplatin-sensitive and cisplatin-resistant cell lines. The primary objectives of this study are to determine the nature of the interactions between BT and cisplatin and to understand the mechanism(s) of action of BT-cisplatin combination. METHODS: The cytotoxic effects of drugs either alone or in combination were evaluated using presto-blue assay. Cellular reactive oxygen species were measured by flow cytometry. Immunoblot analysis was carried out to investigate changes in levels of cleaved PARP, XIAP, bcl-2, bcl-xL, p21 and p27. Luminescent and colorimetric assays were used to test caspases 3/7 and ATX activity. RESULTS: The efficacy of the BT-cisplatin combination depends upon the cell type and concentrations of cisplatin and BT. In cisplatin-sensitive cell lines, BT and cisplatin were mostly antagonistic except when used at low concentrations, where synergy was observed. In contrast, in cisplatin-resistant cells, BT-cisplatin combination treatment displayed synergistic effects at most of the drug ratios/concentrations. Our results further revealed that the synergistic interaction was linked to increased reactive oxygen species generation and apoptosis. Enhanced apoptosis was correlated with loss of pro-survival factors (XIAP, bcl-2, bcl-xL), expression of pro-apoptotic markers (caspases 3/7, PARP cleavage) and enhanced cell cycle regulators p21 and p27. CONCLUSION: In cisplatin-resistant cell lines, BT potentiated cisplatin-induced cytotoxicity at most drug ratios via enhanced ROS generation and modulation of key regulators of apoptosis. Low doses of BT and cisplatin enhanced efficiency of cisplatin treatment in all the ovarian cancer cell lines tested. Our results suggest that novel combinations such as BT and cisplatin might be an attractive therapeutic approach to enhance ovarian cancer chemosensitivity. Combining low doses of cisplatin with subtherapeutic doses of BT can ultimately lead to the development of an innovative combination therapy to reduce/prevent the side effects normally occurring when high doses of cisplatin are administered.

Identification of Multiple Cryptococcal Fungicidal Drug Targets by Combined Gene Dosing and Drug Affinity Responsive Target Stability Screening.

UNLABELLED: Cryptococcus neoformans is a pathogenic fungus that is responsible for up to half a million cases of meningitis globally, especially in immunocompromised individuals. Common fungistatic drugs, such as fluconazole, are less toxic for patients but have low efficacy for initial therapy of the disease. Effective therapy against the disease is provided by the fungicidal drug amphotericin B; however, due to its high toxicity and the difficulty in administering its intravenous formulation, it is imperative to find new therapies targeting the fungus. The antiparasitic drug bithionol has been recently identified as having potent fungicidal activity. In this study, we used a combined gene dosing and drug affinity responsive target stability (GD-DARTS) screen as well as protein modeling to identify a common drug binding site of bithionol within multiple NAD-dependent dehydrogenase drug targets. This combination genetic and proteomic method thus provides a powerful method for identifying novel fungicidal drug targets for further development. IMPORTANCE: Cryptococcosis is a neglected fungal meningitis that causes approximately half a million deaths annually. The most effective antifungal agent, amphotericin B, was developed in the 1950s, and no effective medicine has been developed for this disease since that time. A key aspect of amphotericin B's effectiveness is thought to be because of its ability to kill the fungus (fungicidal activity), rather than just stop or slow its growth. The present study utilized a recently identified fungicidal agent, bithionol, to identify potential fungicidal drug targets that can be used in developing modern fungicidal agents. A combined protein and genetic analysis approach was used to identify a class of enzymes, dehydrogenases, that the fungus uses to maintain homeostasis with regard to sugar nutrients. Similarities in the drug target site were found that resulted in simultaneous inhibition and killing of the fungus by bithionol. These studies thus identify a common, multitarget site for antifungal development.

Stimulation of Slack K(+) Channels Alters Mass at the Plasma Membrane by Triggering Dissociation of a Phosphatase-Regulatory Complex.

Human mutations in the cytoplasmic C-terminal domain of Slack sodium-activated potassium (KNa) channels result in childhood epilepsy with severe intellectual disability. Slack currents can be increased by pharmacological activators or by phosphorylation of a Slack C-terminal residue by protein kinase C. Using an optical biosensor assay, we find that Slack channel stimulation in neurons or transfected cells produces loss of mass near the plasma membrane. Slack mutants associated with intellectual disability fail to trigger any change in mass. The loss of mass results from the dissociation of the protein phosphatase 1 (PP1) targeting protein, Phactr-1, from the channel. Phactr1 dissociation is specific to wild-type Slack channels and is not observed when related potassium channels are stimulated. Our findings suggest that Slack channels are coupled to cytoplasmic signaling pathways and that dysregulation of this coupling may trigger the aberrant intellectual development associated with specific childhood epilepsies.

Broad Bandgap D-A Copolymer Based on Bithiazole Acceptor Unit for Application in High-Performance Polymer Solar Cells with Lower Fullerene Content.

A new broad bandgap and 2D-conjugated D-A copolymer, PBDTBTz-T, based on bithienyl-benzodithiophene donor unit and bithiazole (BTz) acceptor unit, is designed and synthesized for the application as donor material in polymer solar cells (PSCs). The polymer possesses highly coplanar and crystalline structure with a higher hole mobility and lower HOMO energy level which is beneficial to achieve higher open circuit voltage (Voc ) of the PSCs with the polymer as donor. The PSCs based on PBDTBTz-T:PC71 BM blend film with a lower PC71 BM content of 40% demonstrate a power conversion efficiency (PCE) of 6.09% with a relatively higher Voc of 0.92 V. These results indicate that the lower HOMO energy level of the BTz-based D-A copolymer is beneficial to a high Voc of the PSCs. The polymer, with highly coplanar and crystalline structure, can effectively reduce the content of fullerene acceptor in the active layer and can enhance the absorption and PCE of the PSCs.