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

在哺乳动物中，第二信使cAMP由九种跨膜腺苷酸环化酶（tmAC）和一种可溶性AC（sAC）产生。它们都属于普遍存在的核苷酸环化酶III类，其由保守的催化核心定义。然而，sAC催化核心（sAC-cat）活性仅由钙和代谢物ATP和碳酸氢盐刺激。全长sAC还包含一个很难表征的sAC特异性~1100个残基的C末端区（CTR），可能介导其他调节机制。sAC有助于各种生理功能，从神经细胞生长到胰岛素释放，并且被认为是皮肤癌和代谢疾病的治疗靶点。我们研究sAC通过生理和药理学配体及其CTR结构域调节的分子机制，以了解细胞sAC功能，并利用这些机制开发调节剂。在上一个资助期，我们解决了sAC-cat及其与底物，产物，活化剂碳酸氢盐和抑制剂的复合物的第一个晶体结构。他们揭示了sAC催化及其通过碳酸氢盐和利用碳酸氢盐结合位点（BBS）的抑制剂（特别是新发现的变构抑制剂LRE 1）的调节的见解。我们现在开始了两种结构辅助方法，用于改善LRE 1相关抑制剂，并用于开发sAC激活剂和碳酸氢盐依赖性激活的阻断剂（碳酸氢盐阻断剂）。首先，我们进行对接筛选，以确定新的BBS配体不同的sAC状态作为调制器的候选人。其次，我们使用sAC配合物设计潜在的改进化合物衍生物。我们将通过活性和结合研究，并通过解决其sAC复合物的晶体结构来表征这些化合物。机制的见解将指导进一步的开发周期，有前途的化合物将在生理系统中进行表征。使用这种方法，我们已经确定了第一个碳酸氢盐阻滞剂和有希望的候选人sAC激活和抑制。我们进一步开始表征sAC CTR，以获得sAC调节的新见解和用于调节的新靶位点。我们鉴定了来自人sAC和分枝杆菌同源物的推定CTR结构域的第一可溶性表达构建体。我们计划确定其他的，并将这些CTR结构域的结构和生化特征：结构将揭示与已知功能的蛋白质的相似性，结合和活性测定将确定与sAC-cat和小分子配体的潜在相互作用以及CTR结构域及其复合物对sAC活性的影响。我们进一步计划寻找与CTR结构域相互作用的蛋白质，并将通过诱变测试对sAC调控的见解。总之，该项目有望提高我们对sAC和cAMP信号传导的理解，并为生理学研究和药物开发提供化合物。

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.