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 X-ray co-crystallography to split this dual activity, leading to a potent and molecularly efficient RXR agonist, and a first-of-kind inhibitor selective for the RXR/coactivator interaction. Our findings justify future exploration of natural products at dynamic protein interfaces.

Designed Spiroketal Protein Modulation

“…We profiled the RXRα‐activity of (±)‐ 1 alongside full agonist LG100268 (Figure A) in a fluorescence‐based cofactor recruitment assay (Figure , left and Table ). As expected, LG100268 induced potent recruitment of the D22 peptide with an EC 50 =0.10±0.01 μ m . Intriguingly, (±)‐ 1 was also active, with an EC 50 =0.73±0.06 μ m , and additionally exhibited a partial agonist behavior, as judged by the levelling off of polarization at 53 % of the maximum response induced by LG100268 .…”

“…Despite the fact that RXR ligands have been thoroughly investigated, only a very few RXR partial agonists with limited structural diversity have been characterized, and rules guiding the design of heterodimer‐specific RXR ligands are essentially non‐existent. In part as a response to these challenges, and in continuation of our group's recent efforts to identify selective RXR and other NR modulators, we report the first designed spiroketal protein modulator, as exemplified by RXR modulation.…”

“…To evaluate 1 under more biologically relevant conditions, we compared (±)‐ 1 and LG100268 in a mammalian two‐hybrid (M2H) luciferase assay (Figure , right). As expected, LG100268 produced a full and potent concentration‐dependent response (Figure , right). (±)‐ 1 was also active under the assay conditions, though less potent than LG100268 , and the partial response less emphatic than that observed in the FP assay.…”

“…c) Superimposition of R ‐ 1 and final 2 F o − F c electron density map (contoured at 1σ). d) Superimposition of the LBP region of hRXRα bound to R ‐ 1 (protein in green, ligand in orange, PDB code: 5LYQ) and a documented full agonist (protein in red, ligand in cyan, PDB code: 40C7) . The TIF2 peptide corresponding to PDB code 40C7 is shown in cyan.…”

Design, synthesis and conformational analyses of bifacial benzamide based foldamers

“…1b). 31 Direct inhibition of the receptor/co-activator protein-protein interaction, 32-37 notably using helix mimetics 35,36,38 is of potential therapeutic interest as an alternative to the use of competitive inhibitors for the ligand binding site. 39 Herein, we introduce two bifacial proteomimetic scaffolds; bis-benzamide and N-(4-aminophenyl)terephthalamidic as novel foldamers designed as tools to (a) enhance our understanding of aromatic oligoamide foldamer conformation and (b) ligands that could mimic the key side chains at i, i+3, i+4 positions of -helices that participate in PPIs mediated by such a side chain constellation.…”