Click-to-lead design of a picomolar ABA receptor antagonist with potent activity in vivo

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.