Molecular mechanisms regulating the metabolic sensor soluble adenylyl cyclase and development of specific pharmacological modulators

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In mammals, the second messenger cAMP is generated by nine trans-membrane adenylyl cyclases (tmAC) and one soluble AC (sAC). They all belong to the ubiquitous nucleotidyl cyclase Class III, which is defined by a conserved catalytic core. However, sAC catalytic core (sAC-cat) activity is uniquely stimulated by calcium and the metabolites ATP and bicarbonate. Full-length sAC further comprises a hardly characterized, sAC-specific ~1100 residues C-terminal region (CTR) that likely mediates additional regulation mechanisms. sAC contributes to various physiological functions, from nerve cell growth to insulin release, and is considered a therapeutic target for skin cancer and metabolic diseases.We study molecular mechanisms of sAC regulation by physiological and pharmacological ligands and by its CTR domains to understand cellular sAC functions and to exploit these mechanisms for modulator development. In the previous funding period, we solved first crystal structures of sAC-cat and its complexes with substrate, products, the activator bicarbonate, and inhibitors. They revealed insights in sAC catalysis and its regulation by bicarbonate and by inhibitors exploiting the bicarbonate binding site (BBS), in particular the newly discovered, allosteric inhibitor LRE1. We now started two structure-assisted approaches for improvement of LRE1-related inhibitors and for development of sAC activators and blockers for bicarbonate-dependent activation (bicarbonate blockers). First, we perform docking screens to identify novel BBS ligands for different sAC states as modulator candidates. Second, we use sAC complexes for designing potentially improved compound derivatives. We will characterize these compounds through activity and binding studies and by solving crystal structures of their sAC complexes. Mechanistic insights will guide further development cycles, and promising compounds will be characterized in physiological systems. Using this approach we already identified first bicarbonate blockers and promising candidates for sAC activation and inhibition. We further started to characterize the sAC CTR to obtain new insights in sAC regulation and novel target sites for modulation. We identified first soluble expression constructs for putative CTR domains from human sAC and a mycobacterial homolog. We plan to identify additional ones and will characterize these CTR domains structurally and biochemically: Structures will reveal similarities to proteins with known functions, and binding and activity assays will identify potential interactions with sAC-cat and small-molecule ligands and effects of CTR domains and their complexes on sAC activity. We further plan to search for proteins that interact with CTR domains, and we will test insights in sAC regulation via mutagenesis. Taken together, the project promises to improve our understanding of sAC and cAMP signaling and to provide compounds for physiological studies and drug development.