Defining and Exploiting Hypersensitivity Hotspots to Facilitate Abscisic Acid Agonist Optimization

Abstract

Pyrabactin resistance 1 (PYR1) and related abscisic acid (ABA) receptors are new targets for manipulating plant drought tolerance. Here, we identify and use PYR1 hypersensitive mutants to define ligand binding hotspots and show that these can guide improvements in agonist potency. One hotspot residue defined, A160, is part of a pocket that is occupied by ABA’s C6 methyl or by the toluyl methyl of the synthetic agonist quinabactin (QB). A series of QB analogues substituted at the toluyl position were synthesized and provide up to 10-fold gain in activity in vitro. Furthermore, we demonstrate that hypersensitive receptors can be used to improve the sensitivity of a previously described mammalian cell ABA-regulated transcriptional circuit by three orders of magnitude. Collectively, our data show that the systematic mapping of hypersensitivity sites in a ligand-binding pocket can help guide ligand optimization and tune the sensitivity of engineered receptors.

Synthesis and Exploration of Abscisic Acid Receptor Agonists Against Dought Stress by Adding Constraint to a Tetrahydroquinoline‐Based Lead Structure

“…Besides, 2 nd generation lead structure quinabactin 5 a, an oxo-tetrahydroquinolinyl sulfonamide, and its close analogues 5 b-d carrying different N-substituents, or analogues bearing improved phenyl substituents (6 a), a modified oxo-tetrahydroquinoline moiety (6 c) have all shown enhanced in vitro activity paired with significant in vivo efficacy. [29][30][31][32][33][34] In our view these observations foreshow that analogues of plant hormone ABA 1 with considerable structural changes can be identified. Recently, 3 rd generation sulfonamides that show promising receptor binding, as well as significant in vivo efficacy in crops have been identified as lead structures via a target-based high throughput screening (HTS) approach, e. g. phenylsulfonyl ethylenediamines 7 a-b, [35] and via target-based design, e. g. sulfonamide 8, both carrying p-cyano aniline groups.…”

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

“…S8 ). ANT’s carboxylate forms a salt bridge to K56, makes extensive water-mediated contacts with polar residues lining PYL10’s ligand-binding pocket and hydrophobic interactions with the C6 tunnel (which normally accommodates ABA’s C6 methyl and modulates ABA binding affinity) ( 23 ) ( SI Appendix , Fig. S8 G ).…”

“…Chemical modulators of ABA perception have been sought as both research tools for dissecting ABA's role in plant physiology and for their potential agricultural utility (6,7). Dozens of ABA receptor agonists, which reduce transpiration and water use by inducing guard cell closure, have been developed and are being explored as chemical tools for mitigating the effects of drought on crop yields (7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23), most of them either being analogs of ABA or sulfonamides similar to quinabactin (24). ABA receptor antagonists could conceivably be useful in cases where water is not limiting, for example, to increase transpiration and gas exchange under elevated CO 2 in glasshouse agriculture, as germination stimulators, and for studying the ABA dependence of physiological processes, among other applications (25)(26)(27)(28)(29)(30)(31).…”

Dynamic control of plant water use using designed ABA receptor agonists

“…3A), additionally stabilize 3CB-PYL10 interactions. 3CB's cyclohexyl ring packs against L159, V156, Y116, and I106, which line the C6 cleft, a small hydrophobic pocket that normally interacts with ABA's C6 methyl and is a ligand-binding hotspot (47).…”