Classical uncompetitive inhibitors are potent pharmacological modulators of enzyme function. Since they selectively target enzyme-substrate complexes (E:S), their inhibitory potency is amplified by increasing substrate concentrations. Recently, an unconventional uncompetitive inhibitor, called CE3F4R, was discovered for the exchange protein activated by cAMP isoform 1 (EPAC1). Unlike conventional uncompetitive inhibitors, CE3F4R is uncompetitive with respect to an allosteric effector, cAMP, as opposed to the substrate (i.e., CE3F4R targets the E:cAMP rather than the E:S complex). However, the mechanism of CE3F4R as an uncompetitive inhibitor is currently unknown. Here, we elucidate the mechanism of CE3F4R's action using NMR spectroscopy. Due to limited solubility and line broadening, which pose major challenges for traditional structural determination approaches, we resorted to a combination of protein- and ligand-based NMR experiments to comparatively analyze EPAC mutations, inhibitor analogs, and cyclic nucleotide derivatives that trap EPAC at different stages of activation. We discovered that CE3F4R binds within the EPAC cAMP-binding domain (CBD) at a subdomain interface distinct from the cAMP binding site, acting as a wedge that stabilizes a cAMP-bound mixed-intermediate. The mixed-intermediate includes attributes of both the apo/inactive and cAMP-bound/active states. In particular, the intermediate targeted by CE3F4R traps a CBD's hinge helix in its inactive conformation, locking EPAC into a closed domain topology that restricts substrate access to the catalytic domain. The proposed mechanism of action also explains the isoform selectivity of CE3F4R in terms of a single EPAC1 versus EPAC2 amino acid difference that destabilizes the active conformation of the hinge helix.

中文翻译：

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通过变构激动剂的非竞争性调节选择性酶抑制的机制

经典的非竞争性抑制剂是酶功能的有效药理学调节剂。由于它们选择性地靶向酶-底物复合物 (E:S)，因此通过增加底物浓度来增强它们的抑制效力。最近，发现了一种名为 CE3F4R 的非常规非竞争性抑制剂，用于由 cAMP 同种型 1 (EPAC1) 激活的交换蛋白。与传统的非竞争性抑制剂不同，CE3F4R 相对于变构效应物 cAMP 而言是非竞争性的，而不是底物（即，CE3F4R 靶向 E:cAMP 而不是 E:S 复合物）。然而，目前尚不清楚 CE3F4R 作为非竞争性抑制剂的机制。在这里，我们使用核磁共振光谱阐明了 CE3F4R 的作用机制。由于有限的溶解度和谱线加宽，这对传统的结构测定方法构成了重大挑战，我们结合基于蛋白质和配体的 NMR 实验来比较分析 EPAC 突变、抑制剂类似物和在不同激活阶段捕获 EPAC 的环核苷酸衍生物。我们发现 CE3F4R 在与 cAMP 结合位点不同的子域界面处结合在 EPAC cAMP 结合域 (CBD) 内，充当稳定 cAMP 结合的混合中间体的楔子。混合中间体包括 apo/非活性和 cAMP 结合/活性状态的属性。特别是，CE3F4R 靶向的中间体在其非活性构象中捕获 CBD 的铰链螺旋，将 EPAC 锁定到封闭域拓扑结构中，从而限制底物进入催化域。