A Natural‐Product Switch for a Dynamic Protein Interface

Abstract

Small ligands are a powerful way to control the function of protein complexes via dynamic binding interfaces. The classic example is found in gene transcription where small ligands regulate nuclear receptor binding to coactivator proteins via the dynamic activation function 2 (AF2) interface. Current ligands target the ligand-binding pocket side of the AF2. Few ligands are known, which selectively target the coactivator side of the AF2, or which can be selectively switched from one side of the interface to the other. We use NMR spectroscopy and modeling to identify a natural product, which targets the retinoid X receptor (RXR) at both sides of the AF2. We then use chemical synthesis, cellular screening and Xray co-crystallography to split this dual activity, leading to a potent and molecularly efficient RXR agonist, and a first-ofkind inhibitor selective for the RXR/coactivator interaction. Our findings justify future exploration of natural products at dynamic protein interfaces.Supporting information for this article (synthesis protocols, analytical data for all novel compounds, biochemical and cellular evaluation of compounds, including cofactor recruitment, fluorescence polarization, molecular modeling, STD-NMR, CORCEMA-ST, and X-ray co-crystallography) is available on the WWW under http:// dx.

Ligand Dependent Switch from RXR Homo- to RXR-NURR1 Heterodimerization

“…The synthesis and cocrystal structure of ligand 1 were described previously. 35 The allyl side chain of 1 partly occupies the lipophilic pocket in the ligand binding domain of RXRα, analogously to the tetramethyl-cyclohexene unit found in typical RXR ligands, leading to closure of the ligand binding pocket via repositioning of helix 12 in an agonist conformation and subsequent increased binding toward coactivators. The high binding affinity and low molecular weight of 1 makes it an ideal scaffold to explore modifications targeting the lipophilic pocket ( Table 1 ).…”

“…Four boronic acids or esters were then reacted with 12 using Suzuki coupling (Buchwald-modified) to provide intermediates 13 – 16 in excellent yields (78–99%). 35 , 37 The biaryls 13 – 16 were thereafter demethylated using boron tribromide and hydrolyzed using sodium hydroxide, yielding ligands 2 – 5 in reasonable yields with high purities after a preparative HPLC purification. Molecules 6 and 7 were designed and synthesized to access the contorted conformation necessary for the biaryl ligands to fit within the ligand binding pocket of RXRα.…”

“… The syntheses of 1 and 8 have been previously reported. 35 Conditions: (a) thionyl chloride, MeOH, 0 °C; (b) arylboronic acid or arylboronic ester, Pd 2 (dba) 3 , SPhos, KF, dioxane/H 2 O (10:1 v/v), 110 °C; (c) BBr 3 , CH 2 Cl 2 −78 °C; (d) NaOH, dioxane/MeOH (14:5 v/v), 40 °C; (e) bis(pinacolato)diboron, Pd(OAc) 2 , XPhos, KOAc, dioxane 110 °C; (f) 19 or 20 , Pd 2 (dba) 3 , SPhos, KF, dioxane/H 2 O (6:1 v/v), 110 °C; (g) 23 or 24 , Pd 2 (dba) 3 , SPhos, KF, dioxane/H 2 O (6:1 v/v), 110 °C; (h) HCl, THF, room temperature; (i) NaOH, dioxane/MeOH (14:5 v/v). …”

“… 36 Compound 8 ( Figure 1 ) was previously described by us and found to lack significant RXRα activity, because of the additional polar phenolic functionality, which points toward a lipophilic environment. 35 X-ray crystallographic data have demonstrated that alkylation of the appropriate phenol displaces the position of helix 12 toward a (partial) antagonistic fold, influencing the position of L436, which plays a determining role in the communication with helix 12. 36 , 38 Therefore, using the biaryl scaffold, agonist 8 was modified with two different length alkoxy chains.…”

“…The work makes use of the knowledge derived from studies on the natural product 34 honokiol derived RXRα agonists described previously. 35 The biaryl scaffold of this series is straightforward to derivatize, which in principle enables a rapid pharmacophore mapping of the RXRα ligand binding pocket. In this present study, ligands 1 – 5 ( Figure 1 ) were designed to probe the hydrophobic region of the RXRα ligand binding pocket, while keeping the polar interactions intact, to access the flexibility and local displacements of amino acid side-chains of the ligand binding domain.…”