Human soluble adenylyl cyclase (sAC, AC10) - inhibitor (IC50 = 4.0 ± 0.2 μM) [2]

In saltwater, bithionol (0.1–10 mg/mL, 72 h) exhibits toxicity to fresh Paraamoebae parasites [1]. Adenylyl cyclase (AC) activity is inhibited by bithionol (0-100 μM) at IC50 value. Bithionol (50 and 100 μM, 0-10 min) decreases cAMP and almost inhibits sAC. In cells overexpressing 4-4 Cumulative impact, sAC boosts cAMP.

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Enzyme Inhibition Activity: Dose-response experiments on purified human soluble adenylyl cyclase catalytic domains (sAC-cat, residues 1-469) showed that Bithionol potently inhibited sAC activity with an IC50 of 4.0 ± 0.2 μM under assay conditions containing 5 mM ATP, 10 mM MgCl2, and 10 mM CaCl2. It exhibited similar potency to the known sAC inhibitor KH7 and the related compound hexachlorophene (IC50 = 1.6 ± 0.1 μM). [2]
Binding Affinity: Microscale thermophoresis measurements determined the binding affinity (KD) of Bithionol for the apo-sAC protein to be 0.43 ± 0.06 μM, indicating a high-affinity interaction. [2]
Inhibition Mechanism: Kinetic analysis (ATP titrations at varying inhibitor concentrations) revealed that Bithionol acts as a mixed-type inhibitor of sAC. It caused a significant decrease in the apparent Vmax and a weaker increase in the substrate Km, indicating a mostly non-competitive inhibition with respect to ATP, with a small competitive component. Direct fitting with a mixed inhibition model yielded a Ki of 2.3 μM. The inhibition appears to be competitive with the physiological activator bicarbonate, as increasing bicarbonate concentrations shifted the IC50 to higher values (from 6 μM with no bicarbonate to 11 μM with 40 mM bicarbonate). [2]
Structural Basis of Inhibition (X-ray Crystallography): A crystal structure of the human sAC catalytic domains in complex with Bithionol was solved at 2.24 Å resolution. The structure revealed that Bithionol binds deeply into the allosteric bicarbonate binding site (BBS) and occupies the channel connecting this regulatory site to the active site. Key interactions include hydrophobic contacts with Val167, Ala100, Leu162, Leu102, Val175, and a hydrophobic patch formed by Phe338/Phe296, as well as T-shaped π-stacking with Phe45 and Phe336. A hydroxyl group forms a hydrogen bond with the backbone amide of Met337. The chlorine atoms may form polar interactions with Lys95 and backbone atoms. Arg176, a key residue for communication between the active and allosteric sites, interacts with the π-electrons of Bithionol. Binding induces conformational changes, including tightening of the substrate binding site and displacement of the catalytic residue Asp99, which hinders the formation of a productive enzyme-substrate complex. [2]
Mutagenesis Analysis: Inhibition of an sAC-R176A mutant by Bithionol required 2-3 fold higher inhibitor concentrations compared to wild-type sAC, confirming the involvement of Arg176 in inhibitor binding and the inhibition mechanism. [2]
Selectivity over Transmembrane ACs (tmACs): In sAC knockout mouse embryonic fibroblasts (where cAMP production is exclusively from tmACs), Bithionol at concentrations up to 100 μM did not inhibit forskolin-stimulated cAMP accumulation, whereas the tmAC-specific P-site inhibitor 2',5'-dideoxyadenosine did. This demonstrates that Bithionol is specific for sAC relative to tmACs in a cellular context. [2]

In mice, bithionol exhibits moderate efficacy against immature H. nana (100 mg/kg/day, side wall powder for 12 days) [3].

sAC Activity Assay: Activity assays were performed using 100 ng of purified human sAC-cat protein in a buffer containing 50 mM Tris/HCl (pH 8.0), 50 mM NaCl, 10 mM MgCl2, 10 mM CaCl2, and 5 mM ATP. Reactions were incubated at 37°C and stopped by flash freezing. The produced cAMP was quantified using one of three methods: 1) measuring ³²P-labeled cAMP, 2) RapidFire mass spectrometry, or 3) reversed-phase chromatography on a UPLC system with a C18 column using an isocratic mobile phase of 97% 20 mM ammonium acetate (pH 4.5) and 3% acetonitrile. For inhibition studies, compounds were tested at varying concentrations, and data were fitted to appropriate inhibition models. [2]
Binding Assay (Microscale Thermophoresis): The binding affinity of Bithionol for sAC was measured using microscale thermophoresis. A concentration of 0.4 μM sAC-cat protein was used in a buffer containing 50 mM Tris/HCl (pH 8), 50 mM NaCl, and 15 mM CaCl2. The KD value was determined by fitting the binding transition curve with a single-site binding equation. [2]

Cellular cAMP Accumulation Assay (sAC Overexpression): To test the cellular activity of Bithionol, cAMP accumulation was measured in cultured 4-4 cells, which stably overexpress sAC. Cellular phosphodiesterases were inhibited with 500 μM isobutylmethylxanthine. After incubation with the inhibitor for the indicated times, cellular cAMP was quantified using a direct cAMP enzyme immunoassay. Bithionol caused a dose-dependent decrease in cAMP formation, with nearly complete inhibition of sAC-dependent cAMP accumulation observed at 100 μM, similar to the effect of the known sAC inhibitor KH7 (30 μM). [2]
Cellular cAMP Accumulation Assay (tmAC Activity): To assess selectivity, forskolin-stimulated cAMP accumulation was measured in sAC knockout mouse embryonic fibroblasts. Cells were treated with 50 μM forskolin in the presence or absence of inhibitors. Cellular cAMP was quantified as described above. Bithionol at 100 μM did not inhibit this tmAC-dependent cAMP accumulation. [2]

Animal/Disease Models: Immature H. nana infected mice [3]
Doses: 100 mg/kg/day
Route of Administration: Oral administration, 12 days after infection
Experimental Results: 48% of mature H. nana eliminated. LD50: 760 mg/kg.

Absorption, Distribution and Excretion
Bithiophenol is absorbed to a limited extent in the host digestive tract, primarily detected in the blood, especially in bile, and ultimately excreted via bile. Peak drug concentrations in bile are reached within 2 hours of administration. Blood drug concentrations are significantly lower than bile concentrations. When rats were fed (35)S-bithiophenol, (35)S-bithiophenol sulfoxide, or (35)S-bithiophenol sulfone, urinary excretion of the metabolites was very low. Bithiophenol sulfoxide was primarily excreted via feces. Over 90% of the bile radioactivity was present as glucuronides of the three compounds, with over 70% of the glucuronides being bis(thiophenol) glucuronides. Metabolisms/Metabolites Urine, feces, and bile were collected after rats were orally administered (35)S-bis(thiophenol) sulfoxide. Eight metabolites were observed in the urine. Paper chromatography and chemical assays identified one of them as an inorganic sulfate. One strong acid was identified as 3,5-dichloro-2-hydroxysulfonic acid. Two other compounds were identified as bis(thiobisphenol sulfone) and bis(thiobisphenol). In addition, three compounds were identified as bis(thiobisphenol), bis(thiobisphenol sulfoxide), and glucuronide of bis(thiobisphenol sulfone). One metabolite was not identified. Except for free bis(thiobisphenol sulfone), the metabolites found in bile were the same as those found in urine. However, the quantifications differed greatly. Speculum glucuronide of bis(thiobisphenol sulfone) accounted for 71% of (35)S in bile, but only 16.5% in urine. /bis(thiobisphenol sulfoxide/
When rats were fed (35)S-bis(thiobisphenol), (35)S-bis(thiobisphenol sulfoxide), or (35)S-bis(thiobisphenol sulfone), the urinary excretion of the metabolites was…very low. …More than 90% of the bile radioactivity was in the form of glucuronide of these three compounds, of which more than 70% of the glucuronide was in the form of bis(thiobisphenol sulfone) glucuronide. Sulfones are metabolized to produce catechol and guaiacol.
/Thiobisphenol and thiobisphenol sulfone are the main metabolites of thiobisphenol sulfoxide. /

Interactions When thiophene is used in combination with carbon tetrachloride, sodium antimony tartrate, emetine hydrochloride, hexachloroethane, or hexachloro-p-xylene, the toxicity is moderately to significantly enhanced.

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Non-human toxicity values
Oral LD50 in rats: 1430 mg/kg
Oral LD50 in mice: 2100 mg/kg
Intravenous LD50 in mice: 18 mg/kg
General cytotoxicity: The authors noted that they observed general cytotoxicity in thiophene at a concentration of approximately 10 μM, which is the concentration required to significantly inhibit sAC in the cellular environment. [2]
Historical FDA ban: Thiophene was once used as an antimicrobial agent in cosmetics, but due to its photosensitivity, the U.S. Food and Drug Administration (FDA) banned this use. [2]

[1]. In vitro toxicity of bithionol and bithionol sulphoxide to Neoparamoeba spp., the causative agent of amoebic gill disease (AGD). Dis Aquat Organ. 2010 Sep 17;91(3):257-62.

[2]. Bithionol Potently Inhibits Human Soluble Adenylyl Cyclase through Binding to the Allosteric Activator Site. J Biol Chem. 2016 Apr 29;291(18):9776-84.

[3]. Anthelmintic effects of bithionol, paromomycin sulphate, flubendazole and mebendazole on mature and immature Hymenolepis nana in mice. J Helminthol. 1985 Sep;59(3):211-6.

2,2'-Thiobis(4,6-dichlorophenol) is a white or off-white crystalline powder with a very faint aromatic or phenolic odor. (NTP, 1992)
Thiobisphenol is an aryl sulfide, belonging to the diphenyl sulfide family, in which the 2-position of each phenyl group is replaced by a hydroxyl group, and the 3- and 5-positions are replaced by chlorine. It is a fungicide and insecticide, and was once used in various topical medications to treat liver fluke disease, but was withdrawn from the market due to its proven potent photosensitizer, which may cause serious skin diseases. It is currently used as an anti-flatulence drug and antifungal pesticide. It is an aryl sulfide, organochlorine insecticide, dichlorobenzene, polyphenol, bridged diphenyl fungicide, and bridged diphenyl antifungal drug.
Thiophenol was once marketed as an active ingredient in various topical medications, but has been proven to be a potent photosensitizer, which may cause serious skin diseases. On October 24, 1967, the new drug application for bisthiophene was withdrawn (see Federal Register, October 31, 1967 (32 FR 15046)). Bisthiophene is a halogenated anti-infective agent used to treat trematode and tapeworm infections. Mechanism of Action: Bisthiophene interferes with the neuromuscular physiology of worms (the target species), impairs egg formation, and may cause defects in the protective cuticle covering the worms. At the biochemical level, oxidative phosphorylation is inhibited, and the bisthiophene molecule can chelate iron, thereby inactivating iron-containing enzyme systems. In the target species, in vivo treatment of adult worms with bisthiophene reduces glycolysis and oxidative metabolism. Specifically, succinate oxidation is inhibited. Although the exact mechanism of action of bisthiophene is unclear, it is speculated that it may rely on the phenolic hydroxyl group as a hydrogen acceptor, which would normally be involved in reactions associated with succinate oxidation. Interference with these reactions may deprive trematodes of the energy necessary for life.

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Therapeutic Uses
Topical anti-infective agent; anti-flatulence drug
Bismuth subsalicylate is used to treat paragonimiasis at a dose of 30 to 50 mg/kg body weight, orally every other day for 10 to 15 times. The same dose has been used to treat clonorchiasis. Bismuth subsalicylate has also been used to treat fascioliasis at doses up to 3 grams daily, taken every other day for 15 times. In tapeworm infections, the highest dose ever used was 60 mg/kg body weight, divided into two doses approximately one hour apart.
In patients with lung infections, 0.8% develop brain infections. In a group of 24 patients with brain infections, bismuth subsalicylate was effective in all patients, clearing worm eggs from sputum and stopping the production of rusty sputum. However, only 9 patients experienced effective control of brain symptoms after taking the drug, including vision loss, significant meningitis, and one case of intradural abscess.
This drug should be taken with food to reduce the incidence and severity of gastrointestinal symptoms.
For more complete data on the therapeutic uses of BITHIONOL (16 in total), please visit the HSDB record page.

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Background and therapeutic potential: Soluble adenylate cyclase (sAC) is a potential therapeutic target for diseases such as diabetes and glaucoma, and can also be used as a male contraceptive. Bithionol is a novel sAC inhibitor that works through an allosteric mechanism by binding to a physiological activator (bicarbonate). [2]
Mechanism of action: Bithionol is a potent sAC-specific inhibitor that works primarily through a non-competitive allosteric mechanism. It binds to the unique bicarbonate binding site (BBS) of sAC, inducing a conformational change that disrupts the active site and inhibits catalysis. This is the first known sAC inhibitor that acts on this site through this mechanism. [2]
Chemical framework and drug development potential: Although thiophene and its analogue hexachlorophene have pleiotropic biological effects (antiviral, antibacterial, and inhibition of cancer cell growth) and cytotoxicity at effective concentrations, making them unsuitable as drugs, their chemical framework, binding sites, and inhibitory mechanisms provide a valuable starting point for developing more specific and potent sAC-targeted therapies. [2]
Comparison with other inhibitors:
thiophene
binds to BBS more deeply than the weak inhibitor DIDS. Unlike the previously described potent inhibitor ASI-8 (whose action extends from BBS to the substrate binding site),
Bithionol
occupies a regulatory site, resulting in its unique, primarily allosteric inhibitory properties. [2]

C12H6CL4O2S

356.038

353.884

97-18-7

Bithionol (sulfoxide);844-26-8

2406

White to off-white solid powder

1.8±0.1 g/cm3

444.7±45.0 °C at 760 mmHg

188°C

222.8±28.7 °C

0.0±1.1 mmHg at 25°C

1.741

5.51

2

3

2

19

282

0

JFIOVJDNOJYLKP-UHFFFAOYSA-N

InChI=1S/C12H6Cl4O2S/c13-5-1-7(15)11(17)9(3-5)19-10-4-6(14)2-8(16)12(10)18/h1-4,17-18H

2,4-dichloro-6-(3,5-dichloro-2-hydroxyphenyl)sulfanylphenol

Bithionol CP 3438 Bitin CP3438 Lorothidol CP-3438

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ≥ 33 mg/mL (~92.68 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (5.84 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (5.84 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)