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/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.
Highlights
The universal second messenger cAMP regulates various cellular processes such as cell adhesion, gene expression, and energy metabolism [1, 2]
These adenylyl cyclases (ACs) differ in their biological function and regulation. transmembrane AC (tmAC) reside in the plasma membrane and are regulated by heterotrimeric G proteins in response to signals acting via G protein-coupled receptors
Inhibition Is Mostly Non-competitive with ATP—Because HCP is an aggressive substance with pleiotropic effects, potentially including membrane disruption and protein denaturation, we tested whether concentrations of HCP capable of fully inhibiting soluble AC (sAC) affected the structural integrity of the enzyme
Summary
Karyotes, many Class III ACs are active as homodimers of identical domains featuring two symmetric active sites. Only two classes of compounds are known to modulate ACs via the pseudosymmetric regulatory site: forskolin-related compounds, which modulate tmACs and exhibit some isoform selectivity [14], and the sAC inhibitor (4-aminofurazan-3-yl)-[3-(1H-benzoimidazol2-ylmethoxy)phenyl]methanone (ASI-8), which was recently developed by fragment-based screening [20]. In an effort to identify novel scaffolds for the development of isoform-specific AC inhibitors, we tested whether HCP and bithionol (see Fig. 1A), a closely related bioactive organochlorine, might inhibit sAC. We found that both HCP and bithionol are potent inhibitors of human sAC. Biochemical characterization and crystal structure analysis show that bithionol binds to the sAC bicarbonate binding site, revealing new insights in allosteric AC regulation and identifying new leads for drug development
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