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

Abstract

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/CO 2 , 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 sAC⅐bithionol 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 sAC⅐ligand 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.

Functional consequences of a KCNT1 variant associated with status dystonicus and early‐onset infantile encephalopathy

“…Our study leveraged reported, available pharmacologic tools including K Na 1.1 activators (bithionol and loxapine) as well as blockers (quinidine and bepridil) to begin to address the mechanism of gain‐of‐function . Bithionol, an anti‐helminthic drug that additionally activates BK channels, is understood mechanistically to be an allosteric modulator of K Na 1.1 through inhibition of soluble adenylyl cyclase . While K Na 1.1 is thought to be ATP‐insensitive as it lacks the ATP binding domain of its family member KNa 2.1 (encoded by KCNT2) , it is sensitive to divalent cation concentration, which also changes with inhibition of adenylyl cyclase.…”

“…Bithionol, an anti‐helminthic drug that additionally activates BK channels, is understood mechanistically to be an allosteric modulator of K Na 1.1 through inhibition of soluble adenylyl cyclase . While K Na 1.1 is thought to be ATP‐insensitive as it lacks the ATP binding domain of its family member KNa 2.1 (encoded by KCNT2) , it is sensitive to divalent cation concentration, which also changes with inhibition of adenylyl cyclase. Importantly, our study demonstrated that the KCNT1 ‐L437F pathogenic variant is not activated by either compound with presumably different mechanisms of action, whereas current conducted by the wild‐type channel is dramatically increased, suggesting the L437F channel is already maximally conducting, with a high open probability.…”

Discovery of LRE1 as a specific and allosteric inhibitor of soluble adenylyl cyclase

“…However, both KH7 and ASI-8 are long compounds which would extend into the active site and inhibit, at least partially, via competition with the substrate. In fact, bithionol, a cytotoxic organochlorine, was recently found to inhibit sAC by binding to the BBS and this connecting channel 45 , similar to ASI-8. And bithionol indeed inhibits sAC by at least partially competing with substrate ATP.…”

“…In contrast, LRE1 combines high potency and selectivity with stability, solubility and, most importantly, lack of cytotoxicity. In particular, LRE1 exhibits less non-specific toxicity than KH7, the widely used sAC specific inhibitor 2 , 27 , and bithionol, a recently characterized sAC selective inhibitor 45 . Therefore, LRE1 is an ideal candidate to explore the therapeutic possibilities of sAC inhibition.…”

High Efficiency Drug Repurposing for New Antifungal Agents

“…• Bithionol is a halogenated anti-infective agent that is used against trematode and cestode infestations. This drug inhibits human soluble adenylyl cyclase (Kleinboelting et al 2016).…”