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

调节代谢传感器可溶性腺苷酸环化酶的分子机制和特定药理调节剂的开发

• 批准号: 236401975
• 负责人: Professor Dr. Clemens Steegborn
• 金额: --
• 依托单位: Arbeitsgruppe Biochemie I
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2013
• 资助国家: 德国
• 起止时间: 2012-12-31 至 2020-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/236401975?language=en
• 关键词: Molecular; mechanisms; regulating; metabolic; sensor

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.

在哺乳动物中，第二信使cAMP由9个跨膜腺苷酸环化酶(TMAC)和1个可溶性AC(SAC)产生。它们都属于普遍存在的核苷酸环化酶III类，它是由保守的催化核心定义的。然而，SAC催化核心(SAC-CAT)活性唯一地被钙以及代谢物三磷酸腺苷和碳酸氢盐刺激。全长SAC进一步包含一个几乎不具特征性的SAC特异性~1100残基C-末端区域(CTR)，可能介导额外的调节机制。SAC参与多种生理功能，从神经细胞生长到胰岛素释放，被认为是皮肤癌和代谢性疾病的治疗靶点。我们通过生理和药物配体及其CTR结构域来研究SAC调节的分子机制，以了解细胞SAC的功能，并利用这些机制来开发调节剂。在之前的资助期间，我们首先解决了SAC-CAT及其与底物、产物、激活剂碳酸氢钠和抑制剂的络合物的晶体结构。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)的抑制剂进行调节的见解，特别是新发现的变构抑制剂LRE1。我们现在开始了两种结构辅助的方法来改进LRE1相关的抑制剂和开发用于重碳酸盐依赖激活的SAC激活剂和阻断剂(重碳酸盐阻滞剂)。首先，我们进行对接筛选，以确定不同SAC状态的新型BBS配体作为调节剂候选。其次，我们使用SAC络合物来设计潜在改进的化合物衍生物。我们将通过活性和结合研究以及通过求解其SAC络合物的晶体结构来表征这些化合物。机械论的洞察力将指导进一步的开发周期，有希望的化合物将在生理系统中得到表征。使用这种方法，我们已经确定了第一批重碳酸盐阻滞剂和有望激活和抑制SAC的候选药物。我们进一步开始对SAC CTR进行表征，以获得对SAC调控的新见解和新的调控靶点。我们首先从人类SAC和一个分枝杆菌同源物中鉴定出了假定的CTR结构域的可溶表达载体。我们计划确定更多的CTR结构域，并从结构和生化角度对这些结构进行表征：结构将揭示与已知功能的蛋白质的相似之处，结合和活性分析将确定与SAC-CAT和小分子配体的潜在相互作用，以及CTR结构域及其复合体对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，并将通过突变测试SAC调控方面的洞察力。综上所述，该项目有望提高我们对SAC和cAMP信号的理解，并为生理学研究和药物开发提供化合物。