The prototypical second messenger cAMP regulates a wide variety of physiological processes. It can simultaneously mediate diverse functions by acting locally within independently-regulated microdomains. In mammalian cells, two types of adenylyl cyclase generate cAMP; G protein regulated transmembrane adenylyl cyclases and bicarbonate- calcium- and ATP-regulated soluble adenylyl cyclase (sAC). Because each type of cyclase regulates distinct microdomains, understanding cAMP signaling demands methods to distinguish between them. We developed a mass spectrometry based adenylyl cyclase assay which we used to identify a novel sAC-specific inhibitor, LRE1. LRE1 binds to the bicarbonate activator binding site and inhibits sAC via a unique allosteric mechanism. LRE1 prevents sAC-dependent processes in cellular and physiological systems and facilitates exploration of the therapeutic potential of sAC inhibition.

Soluble adenylyl cyclase (sAC) has gained attention as a potential therapeutic target given the role of this enzyme in intracellular signaling. We describe successful efforts to design improved sAC inhibitors amenable for in vivo interrogation of sAC inhibition to assess its potential therapeutic applications. This work culminated in the identification of TDI-10229 (12), which displays nanomolar inhibition of sAC in both biochemical and cellular assays and exhibits mouse pharmacokinetic properties sufficient to warrant its use as an in vivo tool compound.

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.

In mammalian cells, 10 different adenylyl cyclases produce the ubiquitous second messenger, cyclic adenosine monophosphate (cAMP). Amongst these cAMP-generating enzymes, bicarbonate (HCO3−)-regulated soluble adenylyl cyclase (sAC; ADCY10) is uniquely essential in sperm for reproduction. For this reason, sAC has been proposed as a potential therapeutic target for non-hormonal contraceptives for men. Here, we describe key sAC-focused in vitro assays to identify and characterize sAC inhibitors for therapeutic use. The affinity and binding kinetics of an inhibitor can greatly influence in vivo efficacy, therefore, we developed improved assays for assessing these efficacy defining features.

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Endosomal sorting and lysosomal degradation of G‐protein coupled receptors (GPCRs) maintains cell homeostasis by attenuating receptor signaling and desensitizing cells to persistent activation. GPCR lysosomal sorting defects cause aberrant cellular responses that contribute to many diseases, including chronic inflammation and cancer metastasis. Here, we demonstrate that the human α‐arrestin ARRDC3 regulates the lysosomal degradation of protease‐activated receptor‐1 (PAR1), a GPCR for thrombin. PAR1 belongs to a subset of GPCRs, including the purinergic receptor P2Y1, that are sorted to lysosomes independent of receptor ubiquitination. Instead, PAR1 and P2Y1 are targeted to lysosomes by YPXnL motifs that are bound by the ESCRT adaptor protein ALIX. ARRDC3 is required for PAR1 degradation, but not for the degradation of ubiquitinated receptors. ARRDC3 regulates ALIX interaction with PAR1 and the ESCRT‐III protein CHMP4. Intriguingly, depletion of ARRDC3 or ALIX blocks recovery of thrombin‐induced dephosphorylation of Yap1, an effector of the Hippo cell proliferation pathway, suggesting that GPCR degradation is important for regulation of Hippo signaling. This study reveals a novel role for α‐arrestins in the ubiquitin‐independent, ALIX‐mediated endosomal trafficking of GPCRs, and demonstrates the importance of lysosomal sorting in the regulation of GPCR‐mediated proliferative signaling. Grant Funding Source: Supported by NIH NIGMS R01 GM090689