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

Abstract

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.

A Closed Form Model for Molecular Ratchet-Type Chemically Induced Dimerization Modules

“…The sensitivity of molecular ratchets in vitro has been quantified using isothermal calorimetry (ITC), ,, yeast surface display titrations, ELISA-like microplate assays, surface plasmon resonance measurements, , and competitive inhibition IC 50 measurements . In this section, we first derive limiting equations suitable for determining equilibrium dissociation constants using yeast surface display titrations and surface plasmon resonance (SPR)/biolayer interferometry (BLI).…”

“…Because increasing the amount of R relative to PP2C decreases the observed IC 50 values under typical assay conditions (Figure H), small differences in the activity of recombinant proteins from purifications can result in substantial differences in the IC 50 measurement. Thus, it is important to measure the percent active receptor using an independent assay . Our closed form model can also be used to identify the set of K d1 and K d2 values that are consistent with different IC 50 s. Figure I shows isoclines for IC 50 values ranging from 30 to 200 nM under common conditions for the inhibition assay ([PP2C] = 50 nM; [PYR1] = 250 nM).…”

Structure-Based Modulation of the Ligand Sensitivity of a Tomato Dimeric Abscisic Acid Receptor Through a Glu to Asp Mutation in the Latch Loop

“…Our data shows that niacin reverses ABA-mediated HAB1 phosphatase inhibition using SlPYL1 E151D in the sub-micromolar range ( Figure 5C ). However, in comparison to other antagonists, niacin is 100-fold less potent than the best-known ABA antagonist molecule ( Vaidya et al, 2021 ).…”

“…These molecules can modulate ABA signaling dynamically and exogenously either by enhancing drought tolerance or inhibiting ABA mediated responses in plants, respectively, ( Hewage et al, 2020 ; Lozano-Juste et al, 2020 ). Major breakthroughs in this field have been the discovery of Opabactin, a potent ABA agonist for manipulating crop abiotic stress tolerance and water use ( Vaidya et al, 2019 ) and Antabactin, a pan-receptor antagonist that disrupts ABA mediated response in Arabidopsis and different crop species ( Vaidya et al, 2021 ).…”

“…Abscisic acid receptor agonists and antagonists are important tools to achieve either hyper- or hypostimulation of ABA signaling, respectively. Chemical activation of ABA signaling can lead to enhanced drought tolerance ( Cao et al, 2017 ; Vaidya and Cutler, 2022 ) whereas downregulation of ABA signaling -when water sources are not limiting—can enhance crop yield ( Miao et al, 2018 ) or serve to stimulate seed germination ( Vaidya et al, 2021 ).…”