Abscisic acid (ABA) is a key plant hormone that mediates both plant biotic and abiotic stress responses and many other developmental processes. ABA receptor antagonists are useful for dissecting and manipulating ABA’s physiological roles in vivo. We set out to design antagonists that block receptor–PP2C interactions by modifying the agonist opabactin (OP), a synthetically accessible, high-affinity scaffold. Click chemistry was used to create an ∼4,000-member library of C4-diversified opabactin derivatives that were screened for receptor antagonism in vitro. This revealed a peptidotriazole motif shared among hits, which we optimized to yield antabactin (ANT), a pan-receptor antagonist. An X-ray crystal structure of an ANT–PYL10 complex (1.86 Å) reveals that ANT’s peptidotriazole headgroup is positioned to sterically block receptor–PP2C interactions in the 4′ tunnel and stabilizes a noncanonical closed-gate receptor conformer that partially opens to accommodate ANT binding. To facilitate binding-affinity studies using fluorescence polarization, we synthesized TAMRA–ANT. Equilibrium dissociation constants for TAMRA–ANT binding to Arabidopsis receptors range from ∼400 to 1,700 pM. ANT displays improved activity in vivo and disrupts ABA-mediated processes in multiple species. ANT is able to accelerate seed germination in Arabidopsis, tomato, and barley, suggesting that it could be useful as a germination stimulant in species where endogenous ABA signaling limits seed germination. Thus, click-based diversification of a synthetic agonist scaffold allowed us to rapidly develop a high-affinity probe of ABA–receptor function for dissecting and manipulating ABA signaling.

脱落酸 (ABA) 是一种关键的植物激素，可介导植物生物和非生物胁迫反应以及许多其他发育过程。 ABA 受体拮抗剂可用于剖析和操纵 ABA 在体内的生理作用。我们着手通过修饰激动剂奥巴巴汀（OP）（一种合成上可利用的高亲和力支架）来设计阻断受体与 PP2C 相互作用的拮抗剂。点击化学用于创建约 4,000 个成员的 C4 多样化 opabactin 衍生物文库，并在体外筛选其受体拮抗作用。这揭示了命中之间共享的肽三唑基序，我们对其进行了优化以产生抗巴克丁（ANT），一种泛受体拮抗剂。 ANT-PYL10 复合物 (1.86 Å) 的 X 射线晶体结构表明，ANT 的肽三唑头基定位于空间阻断 4' 通道中的受体 - PP2C 相互作用，并稳定非规范的闭门受体构象异构体，该构象异构体部分打开以容纳 ANT绑定。为了促进使用荧光偏振的结合亲和力研究，我们合成了 TAMRA-ANT。 TAMRA-ANT 与拟南芥受体结合的平衡解离常数范围为 ∼400 至 1,700 pM。 ANT 在多个物种中表现出改善的体内活性并破坏 ABA 介导的过程。 ANT 能够加速拟南芥、番茄和大麦的种子发芽，这表明它可以用作内源 ABA 信号传导限制种子发芽的物种的发芽刺激剂。因此，基于点击的合成激动剂支架的多样化使我们能够快速开发 ABA 受体功能的高亲和力探针，用于剖析和操纵 ABA 信号传导。