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.

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.

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.

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.

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.

Bithionol Potently Inhibits Human Soluble Adenylyl Cyclase through Binding to the Allosteric Activator Site.

The signaling molecule cAMP regulates functions ranging from bacterial transcription to mammalian memory. In mammals, cAMP is synthesized by nine transmembrane adenylyl cyclases (ACs) and one soluble AC (sAC). Despite similarities in their catalytic domains, these ACs differ in regulation. Transmembrane ACs respond to G proteins, whereas sAC is uniquely activated by bicarbonate. Via bicarbonate regulation, sAC acts as a physiological sensor for pH/bicarbonate/CO2, and it has been implicated as a therapeutic target, e.g. for diabetes, glaucoma, and a male contraceptive. Here we identify the bisphenols bithionol and hexachlorophene as potent, sAC-specific inhibitors. Inhibition appears mostly non-competitive with the substrate ATP, indicating that they act via an allosteric site. To analyze the interaction details, we solved a crystal structure of an sAC·bithionol complex. The structure reveals that the compounds are selective for sAC because they bind to the sAC-specific, allosteric binding site for the physiological activator bicarbonate. Structural comparison of the bithionol complex with apo-sAC and other sAC·ligand complexes along with mutagenesis experiments reveals an allosteric mechanism of inhibition; the compound induces rearrangements of substrate binding residues and of Arg(176), a trigger between the active site and allosteric site. Our results thus provide 1) novel insights into the communication between allosteric regulatory and active sites, 2) a novel mechanism for sAC inhibition, and 3) pharmacological compounds targeting this allosteric site and utilizing this mode of inhibition. These studies provide support for the future development of sAC-modulating drugs.

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.

Assessment of the antitumor potential of Bithionol in vivo using a xenograft model of ovarian cancer.

In terms of the concept of 'drug repurposing', we focused on pharmaceutical-grade Bithionol (BT) as a therapeutic agent against ovarian cancer. Our recent in-vitro study provides preclinical data suggesting a potential therapeutic role for BT against recurrent ovarian cancer. BT was shown to cause cell death by caspases-mediated apoptosis. The present preliminary study further explores the antitumor potential of pharmaceutical-grade BT in an in-vivo xenograft model of human ovarian cancer. Nude Foxn1 mice bearing SKOV-3 human ovarian tumor xenografts were treated with titrated doses of BT and the therapeutic efficacy of pharmaceutical BT was determined using bioluminescence imaging. BT-induced changes in cell proliferation and apoptosis were evaluated by Ki-67 immunochemical staining and TUNEL assay. The effect of BT on autotaxin levels in serum, ascitic fluid, and tumor tissue was assessed by colorimetric and western blot techniques. BT treatment did not show antitumor potential or enhanced survival time at any of the doses tested. No apparent signs of toxicity were observed with any of the doses tested. Immunohistological analysis of tumor sections did not indicate a significant decrease in cellular proliferation (Ki-67 assay). An increase in apoptosis (by TUNEL assay) was observed in all BT-treated mice compared with vehicle-treated mice. Although BT did not show significant antitumor activity in the present study, the ability of BT to induce apoptosis still makes it a promising therapeutic agent. Further confirmatory and optimization studies are essential to enhance the therapeutic effects of BT.

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.

Novel therapeutic strategy for neurodegeneration by blocking Aß seeding mediated aggregation in models of Alzheimer's disease.

Aß accumulation plays a central role in the pathogenesis of Alzheimer's disease (AD). Recent studies suggest that the process of Aß nucleated polymerization is essential for Aß fibril formation, pathology spreading and toxicity. Therefore, targeting this process represents an effective therapeutic strategy to slow or block disease progression. To discover compounds that might interfere with the Aß seeding capacity, toxicity and pathology spreading, we screened a focused library of FDA-approved drugs in vitro using a seeding polymerization assay and identified small molecule inhibitors that specifically interfered with Aß seeding-mediated fibril growth and toxicity. Mitoxantrone, bithionol and hexachlorophene were found to be the strongest inhibitors of fibril growth and protected primary cortical neuronal cultures against Aß-induced toxicity. Next, we assessed the effects of these three inhibitors in vivo in the mThy1-APPtg mouse model of AD (8-month-old mice). We found that mitoxantrone and bithionol, but not hexachlorophene, stabilized diffuse amyloid plaques, reduced the levels of Aß42 oligomers and ameliorated synapse loss, neuronal damage and astrogliosis. Together, our findings suggest that targeting fibril growth and Aß seeding capacity constitutes a viable and effective strategy for protecting against neurodegeneration and disease progression in AD.

Bithionol inhibits ovarian cancer cell growth in vitro - studies on mechanism(s) of action.

BACKGROUND: Drug resistance is a cause of ovarian cancer recurrence and low overall survival rates. There is a need for more effective treatment approaches because the development of new drug is expensive and time consuming. Alternatively, the concept of 'drug repurposing' is promising. We focused on Bithionol (BT), a clinically approved anti-parasitic drug as an anti-ovarian cancer drug. BT has previously been shown to inhibit solid tumor growth in several preclinical cancer models. A better understanding of the anti-tumor effects and mechanism(s) of action of BT in ovarian cancer cells is essential for further exploring its therapeutic potential against ovarian cancer. METHODS: The cytotoxic effects of BT against a panel of ovarian cancer cell lines were determined by Presto Blue cell viability assay. Markers of apoptosis such as caspases 3/7, cPARP induction, nuclear condensation and mitochondrial transmembrane depolarization were assessed using microscopic, FACS and immunoblotting methods. Mechanism(s) of action of BT such as cell cycle arrest, reactive oxygen species (ROS) generation, autotaxin (ATX) inhibition and effects on MAPK and NF-kB signalling were determined by FACS analysis, immunoblotting and colorimetric methods. RESULTS: BT caused dose dependent cytotoxicity against all ovarian cancer cell lines tested with IC50 values ranging from 19 µM - 60 µM. Cisplatin-resistant variants of A2780 and IGROV-1 have shown almost similar IC50 values compared to their sensitive counterparts. Apoptotic cell death was shown by expression of caspases 3/7, cPARP, loss of mitochondrial potential, nuclear condensation, and up-regulation of p38 and reduced expression of pAkt, pNF-κB, pIκBα, XIAP, bcl-2 and bcl-xl. BT treatment resulted in cell cycle arrest at G1/M phase and increased ROS generation. Treatment with ascorbic acid resulted in partial restoration of cell viability. In addition, dose and time dependent inhibition of ATX was observed. CONCLUSIONS: BT exhibits cytotoxic effects on various ovarian cancer cell lines regardless of their sensitivities to cisplatin. Cell death appears to be via caspases mediated apoptosis. The mechanisms of action appear to be partly via cell cycle arrest, ROS generation and inhibition of ATX. The present study provides preclinical data suggesting a potential therapeutic role for BT against recurrent ovarian cancer.

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.

Involvement of autotaxin/lysophospholipase D expression in intestinal vessels in aggravation of intestinal damage through lymphocyte migration.

Lysophosphatidic acid (LPA) has a critical role in lymphocyte migration to secondary lymphoid organs. Autotaxin (ATX)/lysophospholipase D, in the vascular endothelium, is the main enzyme involved in LPA production. Whether ATX is involved in pathological lymphocyte migration to the inflamed mucosa has not been studied. We investigated the involvement of ATX in inflammatory bowel disease patients and two murine models of colitis. Tissue samples were obtained by intestinal biopsies from patients with Crohn's disease and those with ulcerative colitis with informed consent. ATX immunoreactivity was colocalized with MAdCAM-1-positive high-endothelial-like vessels, close to sites of lymphocyte infiltration. Enhanced expression of ATX mRNA was observed in the inflamed mucosa from Crohn's disease and ulcerative colitis patients. ATX mRNA expression level was remarkably higher in the actively inflamed mucosa than in the quiescent mucosa in the same patient. In the T-cell-transferred mouse model, ATX mRNA expression level gradually increased as colitis developed. In the dextran sodium sulfate mouse model, the expression level was considerably higher in colonic mucosa of chronically developed colitis than in colonic mucosa of acute colitis. Administration of an ATX inhibitor, bithionol, remarkably decreased lymphocyte migration to the intestine and ameliorated both dextran sodium sulfate-induced colitis and CD4-induced ileocolitis. In transwell assays, administration of bithionol or 1-bromo-3(s)-hydroxy-4-(palmitoyloxy) butylphosphonate (BrP-LPA) significantly decreased transmigration of splenocytes through high-endothelial-like vessels induced by TNF-α. We conclude that enhanced expression of ATX in the active mucosa has been implicated in the pathophysiology of inflammatory bowel disease through enhancing aberrant lymphocyte migration to the inflamed mucosa.

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.

Novel inhibitors complexed with glutamate dehydrogenase: allosteric regulation by control of protein dynamics.

Mammalian glutamate dehydrogenase (GDH) is a homohexameric enzyme that catalyzes the reversible oxidative deamination of l-glutamate to 2-oxoglutarate using NAD(P)(+) as coenzyme. Unlike its counterparts from other animal kingdoms, mammalian GDH is regulated by a host of ligands. The recently discovered hyperinsulinism/hyperammonemia disorder showed that the loss of allosteric inhibition of GDH by GTP causes excessive secretion of insulin. Subsequent studies demonstrated that wild-type and hyperinsulinemia/hyperammonemia forms of GDH are inhibited by the green tea polyphenols, epigallocatechin gallate and epicatechin gallate. This was followed by high throughput studies that identified more stable inhibitors, including hexachlorophene, GW5074, and bithionol. Shown here are the structures of GDH complexed with these three compounds. Hexachlorophene forms a ring around the internal cavity in GDH through aromatic stacking interactions between the drug and GDH as well as between the drug molecules themselves. In contrast, GW5074 and bithionol both bind as pairs of stacked compounds at hexameric 2-fold axes between the dimers of subunits. The internal core of GDH contracts when the catalytic cleft closes during enzymatic turnover. None of the drugs cause conformational changes in the contact residues, but all bind to key interfaces involved in this contraction process. Therefore, it seems likely that the drugs inhibit enzymatic turnover by inhibiting this transition. Indeed, this expansion/contraction process may play a major role in the inter-subunit communication and allosteric regulation observed in GDH.